Analysis of Nuclear Reconstitution, Nuclear Envelope Assembly, and Nuclear Pore Assembly Using Xenopus In Vitro Assays

A key event in nuclear formation is the assembly of functional nuclear pores. We have used a nuclear reconstitution system derived from Xenopus eggs to examine the process of nuclear pore assembly in vitro. With this system, we have identified three reagents which interfere with nuclear pore assembly, NEM, GTP gamma S, and the Ca++ chelator, BAPTA. These reagents have allowed us to determine that the assembly of a nuclear pore requires the prior assembly of a double nuclear membrane. Inhibition of nuclear vesicle fusion by pretreatment of the membrane vesicle fraction with NEM blocks pore complex assembly. In contrast, NEM treatment of already fused double nuclear membranes does not block pore assembly. This indicates that NEM inhibits a single step in pore assembly--the initial fusion of vesicles required to form a double nuclear membrane. The presence of GTP gamma S blocks pore assembly at two distinct steps, first by preventing fusion between nuclear vesicles, and second by blocking a step in pore assembly that occurs on already fused double nuclear membranes. Interestingly, when the Ca2+ chelator BAPTA is added to a nuclear assembly reaction, it only transiently blocks nuclear vesicle fusion, but completely blocks nuclear pore assembly. This results in the formation of a nucleus surrounded by a double nuclear membrane, but devoid of nuclear pores. To order the positions at which GTP gamma S and BAPTA interfere with pore assembly, a novel anchored nuclear assembly assay was developed. This assay revealed that the BAPTA-sensitive step in pore assembly occurs after the second GTP gamma S-sensitive step. Thus, through use of an in vitro nuclear reconstitution system, it has been possible to biochemically define and order multiple steps in nuclear pore assembly.

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A distinct vesicle population targets membranes and pore complexes to the nuclear envelope in Xenopus eggs.

The Journal of Cell Biology ◽

10.1083/jcb.112.4.545 ◽

1991 ◽

Vol 112 (4) ◽

pp. 545-556 ◽

Cited By ~ 94

Author(s):

G P Vigers ◽

M J Lohka

Keyword(s):

Nuclear Envelope ◽

Gradient Centrifugation ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Nuclear Envelope Assembly ◽

Pore Complexes ◽

Envelope Membranes ◽

Alpha Glucosidase ◽

Soluble Components

Extracts from Xenopus eggs capable of nuclear envelope assembly in vitro were fractionated by differential and density gradient centrifugation. Nuclear envelope assembly was found to require soluble components in the cytosol and two distinct particulate fractions, which we have called nuclear envelope precursor fractions A and B (NEP-A and NEP-B). Both NEP-A and NEP-B are sensitive to treatments with trypsin, sodium carbonate, and detergents, but can be distinguished from each other by their sensitivities to high salt and N-ethylmaleimide and by their levels of alpha-glucosidase activity. Vesicles in NEP-B bind to chromatin, whereas those in NEP-A do not. NEP-B may therefore be involved in the targeting of membranes to the surface of the chromatin, whereas NEP-A may provide a pool of vesicles that contributes many of the nuclear envelope membranes. NEP-B may also play a role in the assembly of nuclear pore complexes because the density of nuclear pores in the resulting envelope is dependent on the ratio of NEP-B to NEP-A in the reconstituted extract.

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BAF is required for emerin assembly into the reforming nuclear envelope

Journal of Cell Science ◽

10.1242/jcs.114.24.4575 ◽

2001 ◽

Vol 114 (24) ◽

pp. 4575-4585 ◽

Cited By ~ 3

Author(s):

Tokuko Haraguchi ◽

Takako Koujin ◽

Miriam Segura-Totten ◽

Kenneth K. Lee ◽

Yosuke Matsuoka ◽

...

Keyword(s):

Nuclear Envelope ◽

Hela Cells ◽

Core Region ◽

Lamin B ◽

The Core ◽

Double Stranded Dna ◽

Recessive Form ◽

Nuclear Envelope Assembly

Mutations in emerin cause the X-linked recessive form of Emery-Dreifuss muscular dystrophy (EDMD). Emerin localizes at the inner membrane of the nuclear envelope (NE) during interphase, and diffuses into the ER when the NE disassembles during mitosis. We analyzed the recruitment of wildtype and mutant GFP-tagged emerin proteins during nuclear envelope assembly in living HeLa cells. During telophase, emerin accumulates briefly at the ‘core’ region of telophase chromosomes, and later distributes over the entire nuclear rim. Barrier-to-autointegration factor (BAF), a protein that binds nonspecifically to double-stranded DNA in vitro, co-localized with emerin at the ‘core’ region of chromosomes during telophase. An emerin mutant defective for binding to BAF in vitro failed to localize at the ‘core’ in vivo, and subsequently failed to localize at the reformed NE. In HeLa cells that expressed BAF mutant G25E, which did not show ‘core’ localization, the endogenous emerin proteins failed to localize at the ‘core’ region during telophase, and did not assemble into the NE during the subsequent interphase. BAF mutant G25E also dominantly dislocalized LAP2β and lamin A from the NE, but had no effect on the localization of lamin B. We conclude that BAF is required for the assembly of emerin and A-type lamins at the reforming NE during telophase, and may mediate their stability in the subsequent interphase.

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Identification of a soluble precursor complex essential for nuclear pore assembly in vitro

Chromosoma ◽

10.1007/bf00337603 ◽

1990 ◽

Vol 100 (1) ◽

pp. 56-66 ◽

Cited By ~ 54

Author(s):

Marie-Christine Dabauvalle ◽

Karin Loos ◽

Ulrich Scheer

Keyword(s):

Nuclear Pore ◽

Precursor Complex ◽

Nuclear Pore Assembly

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The Interaction of Epac1 and Ran Promotes Rap1 Activation at the Nuclear Envelope

Molecular and Cellular Biology ◽

10.1128/mcb.00242-10 ◽

2010 ◽

Vol 30 (16) ◽

pp. 3956-3969 ◽

Cited By ~ 37

Author(s):

Chang Liu ◽

Maho Takahashi ◽

Yanping Li ◽

Tara J. Dillon ◽

Stefanie Kaech ◽

...

Keyword(s):

Nuclear Envelope ◽

G Protein ◽

Nuclear Pore ◽

Intracellular Camp ◽

Small G Protein ◽

Proliferation And Differentiation ◽

Ras Family ◽

Rap1 Activation

ABSTRACT Epac1 (exchange protein directly activated by cyclic AMP [cAMP]) couples intracellular cAMP to the activation of Rap1, a Ras family GTPase that regulates cell adhesion, proliferation, and differentiation. Using mass spectrometry, we identified the small G protein Ran and Ran binding protein 2 (RanBP2) as potential binding partners of Epac1. Ran is a small G protein best known for its role in nuclear transport and can be found at the nuclear pore through its interaction with RanBP2. Here we demonstrate that Ran-GTP and Epac1 interact with each other in vivo and in vitro. This binding requires a previously uncharacterized Ras association (RA) domain in Epac1. Surprisingly, the interaction of Epac1 with Ran is necessary for the efficient activation of Rap1 by Epac1. We propose that Ran and RanBP2 anchor Epac1 to the nuclear pore, permitting cAMP signals to activate Rap1 at the nuclear envelope.

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Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts.

The Journal of Cell Biology ◽

10.1083/jcb.101.2.518 ◽

1985 ◽

Vol 101 (2) ◽

pp. 518-523 ◽

Cited By ~ 235

Author(s):

M J Lohka ◽

J L Maller

Keyword(s):

Nuclear Envelope ◽

Chromosome Condensation ◽

Sperm Chromatin ◽

Spindle Formation ◽

Nuclear Envelope Breakdown ◽

Multipolar Spindles ◽

Egg Extracts ◽

Nuclear Envelope Assembly ◽

Pronuclear Formation

Incubation of demembranated sperm chromatin in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in nuclear envelope assembly, chromosome decondensation, and sperm pronuclear formation. In contrast, egg extracts made with EGTA-containing buffers induced the sperm chromatin to form chromosomes or irregularly shaped clumps of chromatin that were incorporated into bipolar or multipolar spindles. The 150,000 g supernatants of the EGTA extracts could not alone support these changes in incubated nuclei. However, these supernatants induced not only chromosome condensation and spindle formation, but also nuclear envelope breakdown when added to sperm pronuclei or isolated Xenopus liver or brain nuclei that were incubated in extracts made without EGTA. Similar changes were induced by partially purified preparations of maturation-promoting factor. The addition of calcium chloride to extracts containing condensed chromosomes and spindles caused dissolution of the spindles, decondensation of the chromosomes, and re-formation of interphase nuclei. These results indicate that nuclear envelope breakdown, chromosome condensation, and spindle assembly, as well as the regulation of these processes by Ca2+-sensitive cytoplasmic components, can be studied in vitro using extracts of amphibian eggs.

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A trypsin-sensitive receptor on membrane vesicles is required for nuclear envelope formation in vitro.

The Journal of Cell Biology ◽

10.1083/jcb.107.1.57 ◽

1988 ◽

Vol 107 (1) ◽

pp. 57-68 ◽

Cited By ~ 87

Author(s):

K L Wilson ◽

J Newport

Keyword(s):

Nuclear Envelope ◽

Protein Interactions ◽

Membrane Vesicles ◽

Integral Membrane Protein ◽

Electron Microscopic ◽

Vitro Assay ◽

Nuclear Envelope Assembly ◽

Microscopic Studies ◽

Nuclear Envelope Formation

The reformation of functioning organelles at the end of mitosis presents a problem in vesicle targeting. Using extracts made from Xenopus laevis frog eggs, we have studied in vitro the vesicles that reform the nuclear envelope. In the in vitro assay, nuclear envelope growth is linear with time. Furthermore, the final surface area of the nuclear envelopes formed is directly dependent upon the amount of membrane vesicles added to the assay. Egg membrane vesicles could be fractionated into two populations, only one of which was competent for nuclear envelope assembly. We found that vesicles active in nuclear envelope assembly contained markers (BiP and alpha-glucosidase II) characteristic of the endoplasmic reticulum (ER), but that the majority of ER-derived vesicles do not contribute to nuclear envelope size. This functional distinction between nuclear vesicles and ER-derived vesicles implies that nuclear vesicles are unique and possess at least one factor required for envelope assembly that is lacking in other vesicles. Consistent with this, treatment of vesicles with trypsin destroyed their ability to form a nuclear envelope; electron microscopic studies indicate that the trypsin-sensitive proteins is required for vesicles to bind to chromatin. However, the protease-sensitive component(s) is resistant to treatments that disrupt protein-protein interactions, such as high salt, EDTA, or low ionic strength solutions. We propose that an integral membrane protein, or protein tightly associated with the membrane, is critical for nuclear vesicle targeting or function.

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LEM2 recruits CHMP7 for ESCRT-mediated nuclear envelope closure in fission yeast and human cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1613916114 ◽

2017 ◽

Vol 114 (11) ◽

pp. E2166-E2175 ◽

Cited By ~ 73

Author(s):

Mingyu Gu ◽

Dollie LaJoie ◽

Opal S. Chen ◽

Alexander von Appen ◽

Mark S. Ladinsky ◽

...

Keyword(s):

Nuclear Envelope ◽

Fission Yeast ◽

Nuclear Membrane ◽

Spindle Pole Body ◽

Spindle Pole ◽

Human Cells ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Loss Of Function

Endosomal sorting complexes required for transport III (ESCRT-III) proteins have been implicated in sealing the nuclear envelope in mammals, spindle pole body dynamics in fission yeast, and surveillance of defective nuclear pore complexes in budding yeast. Here, we report that Lem2p (LEM2), a member of the LEM (Lap2-Emerin-Man1) family of inner nuclear membrane proteins, and the ESCRT-II/ESCRT-III hybrid protein Cmp7p (CHMP7), work together to recruit additional ESCRT-III proteins to holes in the nuclear membrane. InSchizosaccharomyces pombe, deletion of the ATPasevps4leads to severe defects in nuclear morphology and integrity. These phenotypes are suppressed by loss-of-function mutations that arise spontaneously inlem2orcmp7, implying that these proteins may function upstream in the same pathway. Building on these genetic interactions, we explored the role of LEM2 during nuclear envelope reformation in human cells. We found that CHMP7 and LEM2 enrich at the same region of the chromatin disk periphery during this window of cell division and that CHMP7 can bind directly to the C-terminal domain of LEM2 in vitro. We further found that, during nuclear envelope formation, recruitment of the ESCRT factors CHMP7, CHMP2A, and IST1/CHMP8 all depend on LEM2 in human cells. We conclude that Lem2p/LEM2 is a conserved nuclear site-specific adaptor that recruits Cmp7p/CHMP7 and downstream ESCRT factors to the nuclear envelope.

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Lamin activity is essential for nuclear envelope assembly in a Drosophila embryo cell-free extract.

The Journal of Cell Biology ◽

10.1083/jcb.119.1.17 ◽

1992 ◽

Vol 119 (1) ◽

pp. 17-25 ◽

Cited By ~ 44

Author(s):

N Ulitzur ◽

A Harel ◽

N Feinstein ◽

Y Gruenbaum

Keyword(s):

Nuclear Envelope ◽

Nuclear Membrane ◽

Membrane Vesicles ◽

Embryo Cell ◽

Drosophila Embryo ◽

Sperm Chromatin ◽

Naked Dna ◽

Nuclear Envelope Assembly

The role of the Drosophila lamin protein in nuclear envelope assembly was studied using a Drosophila in vitro assembly system that reconstitutes nuclei from added sperm chromatin or naked DNA. Upon incubation of the embryonic assembly extract with anti-Drosophila lamin antibodies, the attachment of nuclear membrane vesicles to chromatin surface and nuclear envelope formation did not occur. Lamina assembly and nuclear membrane vesicles attachment to the chromatin were inhibited only when the activity of the 75-kD lamin isoform was inhibited in both soluble and membrane-vesicles fractions. Incubation of decondensed sperm chromatin with an extract that was depleted of nuclear membranes revealed the presence of lamin molecules on the chromatin periphery. In addition, high concentrations of bacterially expressed lamin molecules added to the extract, were able to associate with the chromatin periphery, and did not inhibit nuclear envelope assembly. After nuclear reconstitution, a fraction of the lamin pool was converted into the typical 74- and 76-kD isoforms. Together, these data strongly support an essential role of the lamina in nuclear envelope assembly.

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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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