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 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 Motor-driven motility of fungal nuclear pores organizes chromosomes and fosters nucleocytoplasmic transport

2012 ◽  
Vol 198 (3) ◽  
pp. 343-355 ◽  
Author(s):  
Gero Steinberg ◽  
Martin Schuster ◽  
Ulrike Theisen ◽  
Sreedhar Kilaru ◽  
Andrew Forge ◽  
...  
Keyword(s):  
Nuclear Lamina ◽  
Ustilago Maydis ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nuclear Pores ◽  
Gfp Reporter ◽  
Import And Export ◽  
Reporter Proteins ◽  
Pore Complexes

Exchange between the nucleus and the cytoplasm is controlled by nuclear pore complexes (NPCs). In animals, NPCs are anchored by the nuclear lamina, which ensures their even distribution and proper organization of chromosomes. Fungi do not possess a lamina and how they arrange their chromosomes and NPCs is unknown. Here, we show that motor-driven motility of NPCs organizes the fungal nucleus. In Ustilago maydis, Aspergillus nidulans, and Saccharomyces cerevisiae fluorescently labeled NPCs showed ATP-dependent movements at ∼1.0 µm/s. In S. cerevisiae and U. maydis, NPC motility prevented NPCs from clustering. In budding yeast, NPC motility required F-actin, whereas in U. maydis, microtubules, kinesin-1, and dynein drove pore movements. In the latter, pore clustering resulted in chromatin organization defects and led to a significant reduction in both import and export of GFP reporter proteins. This suggests that fungi constantly rearrange their NPCs and corresponding chromosomes to ensure efficient nuclear transport and thereby overcome the need for a structural lamina.

scholarly journals Piecing together nuclear pore complex assembly during interphase

2009 ◽  
Vol 185 (3) ◽  
pp. 377-379 ◽  
Author(s):  
Michael Rexach
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Supramolecular Structures ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Assembly Process ◽  
Nuclear Pores ◽  
Complex Assembly ◽  
Cell Biol ◽  
Pore Complexes

All nucleocytoplasmic traffic of macromolecules occurs through nuclear pore complexes (NPCs), which function as stents in the nuclear envelope to keep nuclear pores open but gated. Three studies in this issue (Flemming, D., P. Sarges, P. Stelter, A. Hellwig, B. Böttcher, and E. Hurt. 2009. J. Cell Biol. 185:387–395; Makio, T., L.H. Stanton, C.-C. Lin, D.S. Goldfarb, K. Weis, and R.W. Wozniak. 2009. J. Cell Biol. 185:459–491; Onishchenko, E., L.H. Stanton, A.S. Madrid, T. Kieselbach, and K. Weis. 2009. J. Cell Biol. 185:475–491) further our understanding of the NPC assembly process by reporting what happens when the supply lines of key proteins that provide a foundation for building these marvelous supramolecular structures are disrupted.

Nuclear organization of pre-mRNA splicing factors and their substrates

1994 ◽  
Vol 52 ◽  
pp. 18-19
Author(s):  
S. Huang ◽  
T. J. Deerinck ◽  
M. H. Ellisman ◽  
D. L. Spector
Keyword(s):  
Mammalian Cells ◽  
Large Body ◽  
Mrna Splicing ◽  
Electron Microscopic Level ◽  
Splicing Factors ◽  
Electron Microscopic ◽  
Speckled Pattern ◽  
Interchromatin Granule Clusters ◽  
Interchromatin Granule ◽  
Perichromatin Fibrils

Previous studies from our laboratory as well as other laboratories have shown that a variety of pre-mRNA splicing factors are localized to a subnuclear speckled domain when mammalian cells are immunolabeled with antibodies against these pre-mRNA splicing factors. At the electron microscopic level the speckled pattern is composed of both interchromatin granule clusters and perichromatin fibrils. A large body of evidence has accumulated from both our laboratory and other laboratories which has suggested that the perichromatin fibrils represent nascent transcripts and the interchromatin granule clusters represent storage and/or assembly sites for pre-mRNA splicing factors. The majority of substrates for these splicing factors are pre-mRNAs which contain a poly(A) tail of approximately 200-300 nucleotides. During the past year 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 we have found to totally colocalize with pre-mRNA splicing factors at interchromatin granule clusters and perichromatin fibrils.

scholarly journals Dynamic Rearrangement of Nucleoporins during Fungal “Open” Mitosis

2008 ◽  
Vol 19 (3) ◽  
pp. 1230-1240 ◽  
Author(s):  
Ulrike Theisen ◽  
Anne Straube ◽  
Gero Steinberg
Keyword(s):  
Nuclear Envelope ◽  
Protein Import ◽  
Complex Dynamics ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nuclear Pores ◽  
Basic Principles ◽  
Early Anaphase ◽  
Pore Complexes ◽  
Corn Smut

Mitosis in animals starts with the disassembly of the nuclear pore complexes and the breakdown of the nuclear envelope. In contrast to many fungi, the corn smut fungus Ustilago maydis also removes the nuclear envelope. Here, we report on the dynamic behavior of the nucleoporins Nup214, Pom152, Nup133, and Nup107 in this “open” fungal mitosis. In prophase, the nuclear pore complexes disassembled and Nup214 and Pom152 dispersed in the cytoplasm and in the endoplasmic reticulum, respectively. Nup107 and Nup133 initially spread throughout the cytoplasm, but in metaphase and early anaphase occurred on the chromosomes. In anaphase, the Nup107-subcomplex redistributed to the edge of the chromosome masses, where the new envelope was reconstituted. Subsequently, Nup214 and Pom152 are recruited to the nuclear pores and protein import starts. Recruitment of nucleoporins and protein import reached a steady state in G2 phase. Formation of the nuclear envelope and assembly of nuclear pores occurred in the absence of microtubules or F-actin, but not if both were disrupted. Thus, the basic principles of nuclear pore complex dynamics seem to be conserved in organisms displaying open mitosis.

Some electron microscopic studies on intact nuclear 'ghosts' and nuclear membrane fragments

1974 ◽  
Vol 268 (891) ◽  
pp. 109-117 ◽  
Keyword(s):  
Nuclear Membrane ◽  
Single Layer ◽  
Carbon Support ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
External Diameter ◽  
Electron Microscopic ◽  
Optical Images ◽  
Microscopic Studies ◽  
Pore Complexes

The electron optical images of negatively stained intact nuclear ‘ghosts’ are readily identified as flattened and sometimes folded bodies, not unlike those given by mammalian erythrocyte ‘ghosts’. Even at low magnifications ( x 5000) the nuclear pore complexes are clearly revealed and can therefore be used as morphological markers for nuclear membrane. At higher magnifications the nuclear pore complexes are seen to be composed of an eight-sided annulus surrounding a central granule, an inner ring of material, and possibly radial fibrils. Very often fine detail appears to be obscured owing to the fact that there are two layers of double nuclear membrane lying flat on the carbon support film. Occasionally the nuclear ‘ghosts’ appear to be torn apart, in all probability because of the spreading and surface-tension forces applied during the preparation of the negatively stained specimens. In this instance the nuclear pore complexes usually remain intact and the surrounding membrane is disrupted. The nuclear pore complexes are spread as a single layer and greater detail is revealed within the annuli. This is particularly so when partially disrupted nuclear pore complexes are studied. The octagonal annulus appears to be composed of circular macromolecules approximately 20 nm in external diameter with a 5 nm diameter central hole. These macromolecules are linked together and partly masked by other diffuse material. It is proposed that one or more of these macromolecules underlies each of the eight annular subunits. A model for the nuclear pore complex is presented and compared with those proposed by other authors.

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.

Assembly of nuclear pore complexes and annulate lamellae promotes normal pronuclear development in fertilized mammalian oocytes

1998 ◽  
Vol 111 (19) ◽  
pp. 2841-2854 ◽  
Author(s):  
P. Sutovsky ◽  
C. Simerly ◽  
L. Hewitson ◽  
G. Schatten
Keyword(s):  
Cell Types ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Annulate Lamellae ◽  
Oocyte Activation ◽  
Nuclear Pores ◽  
Microtubule Inhibitor ◽  
Cytoplasmic Transport ◽  
Oocyte Cytoplasm ◽  
Pore Complexes

In addition to functional nuclear pore complexes engaged in nucleo-cytoplasmic transport, the cytoplasmic stacks of pore complexes, called annulate lamellae, exist in numerous cell types. Although both annulate lamellae and nuclear pore complexes are present in fertilized mammalian oocytes, their relative roles in the process of fertilization and preimplantation development are not known. Using epifluorescence and electron microscopy, we explored their fate during bovine fertilization. The assembly of annulate lamellae in bovine oocytes was triggered by sperm-oocyte binding and continued concomitantly with the incorporation of the nuclear pores in the nuclear envelopes of the developing male and female pronuclei. This process was also induced by the parthenogenetic activation of metaphase-II-arrested oocytes. Depletion of Ca2+, previously implicated in oocyte activation and in the insertion of pore complexes into the nuclear envelope, prevented the formation of nuclear pore complexes, but not the assembly of annulate lamellae in oocyte cytoplasm. Injection of the nuclear pore antagonist, wheat germ agglutinin, into the cytoplasm of mature oocytes that were subsequently fertilized caused the arrest of pronuclear development, indicating the requirement of nuclear pore complexes for normal pronuclear development. Treatment of the fertilized oocytes with the microtubule inhibitor, nocodazole, prevented gathering of annulate lamellae around the developing pronuclei, insertion of nuclear pores into their nuclear envelopes, and further pronuclear development. The formation of the male pronuclei was reconstituted in Xenopus egg extracts and reflected the behavior of nuclear pores during natural fertilization. These data suggest that nuclear pore complexes are required for normal pronuclear development from its beginning up until pronuclear apposition. Annulate lamellae may be involved in the turnover of nuclear pore complexes during fertilization, which is in turn facilitated by the reorganization of oocyte microtubules and influx of Ca2+ into oocyte cytoplasm.

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.

Sign in / Sign up

Export Citation Format

Share Document