Redistribution of nuclear pores during formation of the redundant nuclear envelope in mouse spermatids

Introduction: We have demonstrated that Glucose-6-phosphatase (G6Pase) activity is localized to the endoplasmic reticulum and nuclear envelope of Mueller glia in the normal and pathological guinea pig retina. Using a combination of this cytochemical technique and high voltage electron microscopy, the distribution of nuclear pores could be clearly observed on the nuclear envelope of Mueller glia because of their anatomical lack of reaction products. This technique was developed to study the three-dimensional structure of nuclei and to calculate total numbers of nuclear pores utilizing a computer graphic analysis system in the normal and pathological retina.Materials and methods: Normal and photocoagulated retina of pigmented adult guinea pigs were perfused with a cold mixture of 0.25% glutaraldehyde and 2% paraformaldehyde in 0.1M cacodylate buffer, and the enucleated globes were hemisected and immersed in the same fixative for 30 min. After sectioning and incubation in the reaction medium for the detection of G6Pase activity by the method of Wachstein-Meisel, the sections were postfixed, dehydrated and embedded in Spurr’s epoxy resin. Serial thick sections (1.0um) were prepared for the observation by a Hitachi high voltage electron microscope (H 1250-M) with an accelerating voltage of 1000 Kv. and pictures were analyzed and three-dimensionally reconstructed by TRI (RATOC Co., Ltd.).

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Faculty Opinions recommendation of Nuclear pores form de novo from both sides of the nuclear envelope.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1032343.373024 ◽

2006 ◽

Author(s):

Brian Burke

Keyword(s):

Nuclear Envelope ◽

De Novo ◽

Nuclear Pores

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Nuclear envelope budding is an evolutionary conserved phenomenon

10.1101/413153 ◽

2018 ◽

Cited By ~ 2

Author(s):

Dimitra Panagaki ◽

Richard Neutze ◽

Johanna L. Höög

Keyword(s):

Nuclear Envelope ◽

Trypanosoma Brucei ◽

Electron Tomography ◽

Human Cell Line ◽

Eukaryotic Cells ◽

Nuclear Pores ◽

Dna Transport ◽

Parasitic Protist ◽

3D Architecture ◽

Cellular Dna

AbstractEukaryotic cells are defined by the compartmentalization of the cytoplasm into organelles, the largest of which is the nucleus, which contains the cellular DNA. Transport into and out of the nucleus is highly regulated and is traditionally thought to occur solely through nuclear pores. However, a small number of papers has repeatedly shown vesicular budding from the nuclear envelopes in different organisms. We used electron microscopy to identify such nuclear envelope budding events in a human cell line,Caenorhabditis elegansworms, the two yeastsSaccharomyces cerevisiaeandSchizosaccharomyces pombeand the parasitic protistTrypanosoma brucei. Progressing to electron tomography, the finer details of the 3D architecture of such budding events was revealed. We summarize all the organisms in which this mode of translocation over the nuclear envelope has been observed and conclude that this may be a fundamental, evolutionary conserved mechanism of transport inside eukaryotic cells.

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The plant nuclear envelope in focus

Biochemical Society Transactions ◽

10.1042/bst0380307 ◽

2010 ◽

Vol 38 (1) ◽

pp. 307-311 ◽

Cited By ~ 13

Author(s):

Katja Graumann ◽

David E. Evans

Keyword(s):

Nuclear Envelope ◽

Recent Progress ◽

Protein Targeting ◽

Nuclear Pores ◽

Associated Proteins ◽

Pore Domain

Recent progress in understanding the plant NE (nuclear envelope) has resulted from significant advances in identifying and characterizing the protein constituents of the membranes and nuclear pores. Here, we review recent findings on the membrane integral and membrane-associated proteins of the key domains of the NE, the pore domain and inner and outer NEs, together with information on protein targeting and NE function.

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Further observations on nucleolar tails in amphibian oocytes

Journal of Cell Science ◽

10.1242/jcs.99.3.515 ◽

1991 ◽

Vol 99 (3) ◽

pp. 515-521

Author(s):

PEDRO LEÓN ◽

JAMES KEZER ◽

ERIC SCHABTACH

Keyword(s):

Electron Microscopy ◽

Nuclear Envelope ◽

Nuclear Membrane ◽

Outer Surface ◽

Light And Electron Microscopy ◽

Nuclear Pores ◽

Core Component ◽

Amphibian Species ◽

Attachment Structures ◽

Convoluted Tubules

Large oocytes from some amphibian species possess beaded or unbeaded intranuclear tails that penetrate the extrachromosomal nucleoli through a distinct pit in their surface and attach to the central core component Here we show, using light and electron microscopy, that tails anchor nucleoli to the nuclear envelope through intricate attachment structures. These structures are composed of interconnected spherical masses containing highly convoluted tubules and associated extratubular proteins, directly directly in contact with the inner nuclear membrane. Fibers emerging from the nuclear pores seemingly hold the attachment complex in place. Beads on the nucleolar tails are formed by the accumulation of proteins on the outer surface of smooth tubules. The function of these intranuclear tubules is unknown

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Sequential degradation of proteins from the nuclear envelope during apoptosis

Journal of Cell Science ◽

10.1242/jcs.114.20.3643 ◽

2001 ◽

Vol 114 (20) ◽

pp. 3643-3653 ◽

Cited By ~ 2

Author(s):

Madeleine Kihlmark ◽

Gabriela Imreh ◽

Einar Hallberg

Keyword(s):

Nuclear Envelope ◽

Apoptotic Cell ◽

Nuclear Periphery ◽

Condensed Chromatin ◽

Nuclear Pore ◽

Dependent Manner ◽

Nuclear Pores ◽

Double Labeling ◽

Lamin B ◽

Nuclear Apoptosis

We have produced new antibodies specific for the integral pore membrane protein POM121. Using these antibodies we show that during apoptosis POM121 becomes proteolytically degraded in a caspase-dependent manner. The POM121 antibodies and antibodies specific for other proteins of the nuclear envelope were used in a comparative study of nuclear apoptosis in staurosporine-treated buffalo rat liver cells. Nuclei from these cells were classified in three different stages of apoptotic progression: stage I, moderately condensed chromatin surrounded by a smooth nuclear periphery; stage II, compact patches of condensed chromatin collapsing against a smooth nuclear periphery; stage III, round compact chromatin bodies surrounded by grape-shaped nuclear periphery. We have performed double labeling immunofluorescence microscopy of individual apoptotic cells and quantitative immunoblotting analysis of total proteins from apoptotic cell cultures. The results showed that degradation of nuclear envelope marker proteins occurred in a specific order. POM121 degradation occurred surprisingly early and was initiated before nucleosomal DNA degradation could be detected using TUNEL assay and completed before clustering of the nuclear pores. POM121 was eliminated significantly more rapid compared with NUP153 (a peripheral protein located in the nucleoplasmic basket of the nuclear pore complex) and lamin B (a component of the nuclear lamina). Disappearance of NUP153 and lamin B was coincident with onset of DNA fragmentation and clustering of nuclear pores. By contrast, the peripheral NPC protein p62 was degraded much later. The results suggest that degradation of POM121 may be an important early step in propagation of nuclear apoptosis.

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Evidence for mediated protein uptake by amphibian oocyte nuclei.

The Journal of Cell Biology ◽

10.1083/jcb.96.5.1486 ◽

1983 ◽

Vol 96 (5) ◽

pp. 1486-1490 ◽

Cited By ~ 34

Author(s):

C M Feldherr ◽

R J Cohen ◽

J A Ogburn

Keyword(s):

Nuclear Envelope ◽

Equilibrium Distribution ◽

Diffusion Rate ◽

Axial Ratio ◽

Nuclear Pores ◽

Simple Diffusion ◽

Protein Uptake ◽

Nuclear Uptake ◽

Nuclear Extracts ◽

Endogenous Proteins

The objective of this investigation was to determine whether there is mediated transport of endogenous proteins across the nuclear envelope. For this purpose, we studied the nuclear uptake of a 148,000-dalton Rana oocyte polypeptide (RN1) and compared its actual uptake rate with the rate that would be expected if RN1 crossed the envelope by simple diffusion through the nuclear pores. Nuclear uptake was studied in two ways: first, oocytes were incubated in L-[3H]leucine for 1 h and, at various intervals after labeling, the amount of 3H-RN1 present in the nucleoplasm was determined. Second, L-[3H]leucine-labeled nuclear extracts, containing RN1, were microinjected into the cytoplasm of nonlabeled cells, and the proportion of 3H-RN1 that subsequently entered the nucleus was measured. It was found that RN1 can readily penetrate the nuclear envelope; for example, after 6 h, approximately 36% of the newly synthesized RN1 and 17% of the injected RN1 had entered the nucleus. The diffusion rate through pores having a radius of 45 A was calculated for several possible molecular configurations of RN1. Using axial ratios of 34, 7.5, 2, and 1, the estimated times required to reach 63% of diffusion equilibrium are 757, 468, 6,940 h, and infinity, respectively. Even assuming an axial ratio of 7.5 (the most diffusive configuration) and an equilibrium distribution of 45, simple diffusion through the pores could account for only approximately 1/20 the observed nuclear uptake of RN1. This and other comparisons indicate that some form of mediated transport is involved in the nucleocytoplasmic exchange of this polypeptide.

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Piecing together nuclear pore complex assembly during interphase

The Journal of Cell Biology ◽

10.1083/jcb.200904022 ◽

2009 ◽

Vol 185 (3) ◽

pp. 377-379 ◽

Cited By ~ 9

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.

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A lamin-independent pathway for nuclear envelope assembly.

The Journal of Cell Biology ◽

10.1083/jcb.111.6.2247 ◽

1990 ◽

Vol 111 (6) ◽

pp. 2247-2259 ◽

Cited By ~ 199

Author(s):

J W Newport ◽

K L Wilson ◽

W G Dunphy

Keyword(s):

Nuclear Envelope ◽

Dna Synthesis ◽

Structural Integrity ◽

Nuclear Lamina ◽

Limited Extent ◽

Nuclear Pores ◽

Nuclear Assembly ◽

Nuclear Envelope Assembly ◽

A Cell ◽

Nuclear Envelope Formation

The nuclear envelope is composed of membranes, nuclear pores, and a nuclear lamina. Using a cell-free nuclear assembly extract derived from Xenopus eggs, we have investigated how these three components interact during nuclear assembly. We find that the Xenopus embryonic lamin protein LIII cannot bind directly to chromatin or membranes when each is present alone, but is readily incorporated into nuclei when both of the components are present together in an assembly extract. We find that depleting lamin LIII from an extract does not prevent formation of an envelope consisting of membranes and nuclear pores. However, these lamin-depleted envelopes are extremely fragile and fail to grow beyond a limited extent. This suggests that lamin assembly is not required during the initial steps of nuclear envelope formation, but is required for later growth and for maintaining the structural integrity of the envelope. We also present results showing that lamins may only be incorporated into nuclei after DNA has been encapsulated within an envelope and nuclear transport has been activated. With respect to nuclear function, our results show that the presence of a nuclear lamina is required for DNA synthesis to occur within assembled nuclei.

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Xenopus lamin B3 has a direct role in the assembly of a replication competent nucleus: evidence from cell-free egg extracts

Journal of Cell Science ◽

10.1242/jcs.108.11.3451 ◽

1995 ◽

Vol 108 (11) ◽

pp. 3451-3461 ◽

Cited By ~ 12

Author(s):

M. Goldberg ◽

H. Jenkins ◽

T. Allen ◽

W.G. Whitfield ◽

C.J. Hutchison

Keyword(s):

Dna Replication ◽

Nuclear Envelope ◽

Nuclear Matrix ◽

Nuclear Pore ◽

Major Protein ◽

Protein Species ◽

Nuclear Pores ◽

Thin Sections ◽

Egg Extracts ◽

Nuclear Envelope Assembly

Xenopus egg extracts which assemble replication competent nuclei in vitro were depleted of lamin B3 using monoclonal antibody L6 5D5 linked to paramagnetic beads. After depletion, the extracts were still capable of assembling nuclei around demembranated sperm heads. Using field emission in lens scanning electron microscopy (FEISEM) we show that most nuclei assembled in lamin B3-depleted extracts have continuous nuclear envelopes and well formed nuclear pores. However, several consistent differences were observed. Most nuclei were small and only attained diameters which were half the size of controls. In a small number of nuclei, nuclear pore baskets, normally present on the inner aspect of the nuclear envelope, appeared on its outer surface. Finally, the assembly of nuclear pores was slower in lamin B3-depleted extracts, indicating a slower overall rate of nuclear envelope assembly. The results of FEISEM were confirmed using conventional TEM thin sections, where again the majority of nuclei assembled in lamin B3-depleted extracts had well formed double unit membranes containing a high density of nuclear pores. Since nuclear envelope assembly was mostly normal but slow in these nuclei, the lamin content of ‘depleted’ extracts was investigated. While lamin B3 was recovered efficiently from cytosolic and membrane fractions by our procedure, a second minor lamin isoform, which has characteristics similar to those of the somatic lamin B2, remained in the extract. Thus it is likely that this lamin is necessary for nuclear envelope assembly. However, while lamin B2 did not co-precipitate with lamin B3 during immunodepletion experiments, several protein species did specifically associate with lamin B3 on paramagnetic immunobeads. The major protein species associated with lamin B3 migrated with molecular masses of 102 kDa and 57 kDa, respectively, on one-dimensional polyacrylamide gels. On two-dimensional O'Farrell gels the mobility of the 102 kDa protein was identical to the mobility of a major nuclear matrix protein, indicating a specific association between lamin B3 and other nuclear matrix proteins. Nuclei assembled in lamin B3-depleted extracts did not assemble a lamina, judged by indirect immunofluorescence, and failed to initiate semi-conservative DNA replication. However, by reinoculating depleted extracts with purified lamin B3, nuclear lamina assembly and DNA replication could both be rescued. Thus it seems likely that the inability of lamin-depleted extracts to assemble a replication competent nucleus is a direct consequence of a failure to assemble a lamina.

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