scholarly journals Mitotic checkpoint proteins HsMAD1 and HsMAD2 are associated with nuclear pore complexes in interphase

2001 ◽  
Vol 114 (5) ◽  
pp. 953-963 ◽  
Author(s):  
M.S. Campbell ◽  
G.K. Chan ◽  
T.J. Yen
Keyword(s):  
Mammalian Cells ◽  
Metaphase Plate ◽  
Mitotic Checkpoint ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Checkpoint Proteins ◽  
Anaphase Onset ◽  
Human Proteins ◽  
Tax Interaction ◽  
Pore Complexes

Mad1 was first identified in budding yeast as an essential component of the checkpoint system that monitors spindle assembly in mitosis and prevents premature anaphase onset. Using antibodies to the human homologue of Mad1 (HsMAD1), we have begun to characterize this protein in mammalian cells. HsMad1 is found localized at kinetochores in mitosis. The labeling is brightest in prometaphase and is absent from kinetochores at metaphase and anaphase. In cells where most chromosomes have reached the metaphase plate, those aligned at the plate show no labeling while remaining, unaligned chromosomes are still brightly labeled. We find HsMad1 associated with HsMad2. Association with p55CDC, a protein previously shown to bind HsMad2, was not detected. Surprisingly, unlike any other known mitotic checkpoint proteins, HsMad1 and HsMAD2 were found localized at nuclear pores throughout interphase. This was confirmed by co-labeling with an antibody to known nuclear pore complex proteins and by their co-purification with enriched nuclear envelope fractions. HsMad1 was identified serendipitously by its binding to a viral protein, HTLV-1 Tax, which affects transcription of viral and human proteins. The localization of HsMad1 to nuclear pore complexes suggests an alternate, non-mitotic role for the Mad1/Tax interaction in the viral transformation of cells.

scholarly journals In Vivo Dynamics of Drosophila Nuclear Envelope Components

2008 ◽  
Vol 19 (9) ◽  
pp. 3652-3666 ◽  
Author(s):  
Katerina R. Katsani ◽  
Roger E. Karess ◽  
Nathalie Dostatni ◽  
Valérie Doye
Keyword(s):  
Nuclear Envelope ◽  
Mammalian Cells ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Checkpoint Proteins ◽  
Early Anaphase ◽  
Associated Proteins ◽  
Pore Complexes ◽  
Drosophila Cells

Nuclear pore complexes (NPCs) are multisubunit protein entities embedded into the nuclear envelope (NE). Here, we examine the in vivo dynamics of the essential Drosophila nucleoporin Nup107 and several other NE-associated proteins during NE and NPCs disassembly and reassembly that take place within each mitosis. During both the rapid mitosis of syncytial embryos and the more conventional mitosis of larval neuroblasts, Nup107 is gradually released from the NE, but it remains partially confined to the nuclear (spindle) region up to late prometaphase, in contrast to nucleoporins detected by wheat germ agglutinin and lamins. We provide evidence that in all Drosophila cells, a structure derived from the NE persists throughout metaphase and early anaphase. Finally, we examined the dynamics of the spindle checkpoint proteins Mad2 and Mad1. During mitotic exit, Mad2 and Mad1 are actively imported back from the cytoplasm into the nucleus after the NE and NPCs have reformed, but they reassociate with the NE only later in G1, concomitantly with the recruitment of the basket nucleoporin Mtor (the Drosophila orthologue of vertebrate Tpr). Surprisingly, Drosophila Nup107 shows no evidence of localization to kinetochores, despite the demonstrated importance of this association in mammalian cells.

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.

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 Control of the spindle checkpoint by lateral kinetochore attachment and limited Mad1 recruitment

2015 ◽  
Vol 26 (14) ◽  
pp. 2620-2639 ◽  
Author(s):  
Nathaniel I. Krefman ◽  
David G. Drubin ◽  
Georjana Barnes
Keyword(s):  
Binding Sites ◽  
De Novo ◽  
Spindle Checkpoint ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Fully Depleted ◽  
Checkpoint Proteins ◽  
Kinetochore Assembly ◽  
Sister Chromatids ◽  
Pore Complexes

We observed the dynamic recruitment of spindle checkpoint proteins Mad1 and Bub1 to detached kinetochores in budding yeast using real-time live-cell imaging and quantified recruitment in fixed cells. After induced de novo kinetochore assembly at one pair of sister centromeres, Mad1 appeared after the kinetochore protein Mtw1. Detached kinetochores were not associated with the nuclear envelope, so Mad1 does not anchor them to nuclear pore complexes (NPCs). Disrupting Mad1's NPC localization increased Mad1 recruitment to detached sister kinetochores. Conversely, increasing the number of detached kinetochores reduced the amount of Mad1 per detached kinetochore. Bub1 also relocalized completely from the spindle to detached sister centromeres after kinetochore assembly. After their capture by microtubules, Mad1 and Bub1 progressively disappeared from kinetochores. Sister chromatids that arrested with a lateral attachment to one microtubule exhibited half the Mad1 of fully detached sisters. We propose that detached kinetochores compete with alternate binding sites in the nucleus to recruit Mad1 and Bub1 from available pools that are small enough to be fully depleted by just one pair of detached kinetochores and that lateral attachment licenses Mad1 removal from kinetochores after a kinetic delay.

scholarly journals In vivo analysis of the stability and transport of nuclear poly(A)+ RNA.

1994 ◽  
Vol 126 (4) ◽  
pp. 877-899 ◽  
Author(s):  
S Huang ◽  
T J Deerinck ◽  
M H Ellisman ◽  
D L Spector
Keyword(s):  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Stable Population ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nuclear Pores ◽  
Electron Microscopic ◽  
Interchromatin Granule Clusters ◽  
Interchromatin Granule ◽  
Pore Complexes

We have studied the distribution of poly(A)+ RNA in the mammalian cell nucleus and its transport through nuclear pores by fluorescence and electron microscopic in situ hybridization. Poly(A)+ RNA was detected in the nucleus as a speckled pattern which includes interchromatin granule clusters and perichromatin fibrils. When cells are fractionated by detergent and salt extraction as well as DNase I digestion, the majority of the nuclear poly(A)+ RNA was found to remain associated with the nonchromatin RNP-enriched fraction of the nucleus. After inhibition of RNA polymerase II transcription for 5-10 h, a stable population of poly(A)+ RNA remained in the nucleus and was reorganized into fewer and larger interchromatin granule clusters along with pre-mRNA splicing factors. This stable population of nuclear RNA may play an important role in nuclear function. Furthermore, we have observed that, in actively transcribing cells, the regions of poly(A)+ RNA which reached the nuclear pore complexes appeared as narrow concentrations of RNA suggesting a limited or directed pathway of movement. All of the observed nuclear pores contained poly(A)+ RNA staining suggesting that they are all capable of exporting RNA. In addition, we have directly visualized, for the first time in mammalian cells, the transport of poly(A)+ RNA through the nuclear pore complexes.

scholarly journals Nup358 integrates nuclear envelope breakdown with kinetochore assembly

2003 ◽  
Vol 162 (6) ◽  
pp. 991-1001 ◽  
Author(s):  
Davide Salina ◽  
Paul Enarson ◽  
J.B. Rattner ◽  
Brian Burke
Keyword(s):  
Nuclear Envelope ◽  
Mitotic Checkpoint ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nuclear Envelope Breakdown ◽  
Chromosome Congression ◽  
Chromatid Segregation ◽  
Pore Complexes ◽  
Membrane Components ◽  
And Function

Nuclear envelope breakdown (NEBD) and release of condensed chromosomes into the cytoplasm are key events in the early stages of mitosis in metazoans. NEBD involves the disassembly of all major structural elements of the nuclear envelope, including nuclear pore complexes (NPCs), and the dispersal of nuclear membrane components. The breakdown process is facilitated by microtubules of the mitotic spindle. After NEBD, engagement of spindle microtubules with chromosome-associated kinetochores leads to chromatid segregation. Several NPC subunits relocate to kinetochores after NEBD. siRNA-mediated depletion of one of these proteins, Nup358, reveals that it is essential for kinetochore function. In the absence of Nup358, chromosome congression and segregation are severely perturbed. At the same time, the assembly of other kinetochore components is strongly inhibited, leading to aberrant kinetochore structure. The implication is that Nup358 plays an essential role in integrating NEBD with kinetochore maturation and function. Mitotic arrest associated with Nup358 depletion further suggests that mitotic checkpoint complexes may remain active at nonkinetochore sites.

scholarly journals Live imaging of single nuclear pores reveals unique assembly kinetics and mechanism in interphase

2010 ◽  
Vol 191 (1) ◽  
pp. 15-22 ◽  
Author(s):  
Elisa Dultz ◽  
Jan Ellenberg
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Constant Rate ◽  
The Reformation ◽  
Assembly Kinetics ◽  
Live Cell Microscopy ◽  
Pore Complexes ◽  
Cell Microscopy

In metazoa, new nuclear pore complexes (NPCs) form at two different cell cycle stages: at the end of mitosis concomitant with the reformation of the nuclear envelope and during interphase. However, the mechanisms of these assembly processes may differ. In this study, we apply high resolution live cell microscopy to analyze the dynamics of single NPCs in living mammalian cells during interphase. We show that nuclear growth and NPC assembly are correlated and occur at a constant rate throughout interphase. By analyzing the kinetics of individual NPC assembly events, we demonstrate that they are initiated by slow accumulation of the membrane nucleoporin Pom121 followed by the more rapid association of the soluble NPC subcomplex Nup107–160. This inverse order of recruitment and the overall much slower kinetics compared with postmitotic NPC assembly support the conclusion that the two processes occur by distinct molecular mechanisms.

scholarly journals Nuclear pore complexes: dynamics in unexpected places

2001 ◽  
Vol 154 (1) ◽  
pp. 17-20 ◽  
Author(s):  
Susan K. Lyman ◽  
Larry Gerace
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Mammalian Cells ◽  
In Vivo Studies ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Pore Complexes ◽  
New Evidence

In vivo studies on the dynamics of the nuclear pore complex (NPC) in yeast suggested that NPCs are highly mobile in the nuclear envelope. However, new evidence indicates that in mammalian cells NPCs are stably attached to a flexible lamina framework, but a peripheral component can exchange rapidly with an intranuclear pool.

scholarly journals The SUMO-specific isopeptidase SENP2 associates dynamically with nuclear pore complexes through interactions with karyopherins and the Nup107-160 nucleoporin subcomplex

2011 ◽  
Vol 22 (24) ◽  
pp. 4868-4882 ◽  
Author(s):  
Jacqueline Goeres ◽  
Pak-Kei Chan ◽  
Debaditya Mukhopadhyay ◽  
Hong Zhang ◽  
Brian Raught ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Mammalian Cells ◽  
Nuclear Export Signal ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Localization Signal ◽  
Import And Export ◽  
Pore Complexes ◽  
Eukaryotic Organisms ◽  
Export Receptors

The association of small, ubiquitin-related modifier–specific isopeptidases (also known as sentrin-specific proteases, or SENPs) with nuclear pore complexes (NPCs) is conserved in eukaryotic organisms ranging from yeast to mammals. However, the functional significance of this association remains poorly understood, particularly in mammalian cells. In this study, we have characterized the molecular basis for interactions between SENP2 and NPCs in human cells. Using fluorescence recovery after photobleaching, we demonstrate that SENP2, although concentrated at the nuclear basket, is dynamically associated with NPCs. This association is mediated by multiple targeting elements within the N-terminus of SENP2 that function cooperatively to mediate NPC localization. One of these elements consists of a high-affinity nuclear localization signal that mediates indirect tethering to FG-repeat–containing nucleoporins through karyopherins. A second element mediates interactions with the Nup107-160 nucleoporin subcomplex. A third element consists of a nuclear export signal. Collectively, our findings reveal that SENP2 is tethered to NPCs through a complex interplay of interactions with nuclear import and export receptors and nucleoporins. Disruption of these interactions enhances SENP2 substrate accessibility, suggesting an important regulatory node in the SUMO pathway.

High resolution analysis of mammalian nuclear structure throughout the cell cycle: implications for nuclear pore complex assembly during interphase and mitosisThis 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. 423-430 ◽  
Author(s):  
Sheona P. Drummond ◽  
Sandra A. Rutherford ◽  
Helen S. Sanderson ◽  
Terry D. Allen
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Molecular Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
High Resolution Analysis ◽  
Multiple Copies ◽  
A Cell ◽  
Specific Proteins ◽  
Pore Complexes

Nuclear pore complexes (NPCs) are the gateways for both active and passive bidirectional molecular transport between the nucleoplasm and cytoplasm. These mega-dalton assemblies are composed of multiple copies of approximately 30 distinct proteins termed nucleoporins. Higher eukaryotes display an “open” mitosis in which the NPCs, nuclear envelope, and lamina disassemble. During mitosis several nucleoporins are redistributed to kinetochores until they are recruited back to the periphery of chromatin as the NPCs are reassembled. Within this study we have developed and optimized the visualization of mammalian cells and their chromosome profiles throughout the cell-cycle. Close attention has been paid to the preservation of chromatin, membranes, and NPC structure to investigate the ultrastructural locations of specific proteins in both interphase and mitosis.

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