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Nuclear envelope breakdown in starfish oocytes proceeds by partial NPC disassembly followed by a rapidly spreading fenestration of nuclear membranes.

Lénárt P, Rabut G, Daigle N, Hand AR, Terasaki M, Ellenberg J - J. Cell Biol. (2003)

Bottom Line: In phase II the NE was completely permeabilized within 35 s.This rapid permeabilization spread as a wave from one epicenter on the animal half across the nuclear surface and allowed free diffusion of particles up to approximately 100 nm in diameter into the nucleus.We conclude that NE breakdown in starfish oocytes is triggered by slow sequential disassembly of the NPCs followed by a rapidly spreading fenestration of the NE caused by the removal of nuclear pores from nuclear membranes still attached to the lamina.

View Article: PubMed Central - PubMed

Affiliation: Gene Expression and Cell Biology/Biophysics Programmes, European Molecular Biology Laboratory, D-69117 Heidelberg, Germany.

ABSTRACT
Breakdown of the nuclear envelope (NE) was analyzed in live starfish oocytes using a size series of fluorescently labeled dextrans, membrane dyes, and GFP-tagged proteins of the nuclear pore complex (NPC) and the nuclear lamina. Permeabilization of the nucleus occurred in two sequential phases. In phase I the NE became increasingly permeable for molecules up to approximately 40 nm in diameter, concurrent with a loss of peripheral nuclear pore components over a time course of 10 min. The NE remained intact on the ultrastructural level during this time. In phase II the NE was completely permeabilized within 35 s. This rapid permeabilization spread as a wave from one epicenter on the animal half across the nuclear surface and allowed free diffusion of particles up to approximately 100 nm in diameter into the nucleus. While the lamina and nuclear membranes appeared intact at the light microscopic level, a fenestration of the NE was clearly visible by electron microscopy in phase II. We conclude that NE breakdown in starfish oocytes is triggered by slow sequential disassembly of the NPCs followed by a rapidly spreading fenestration of the NE caused by the removal of nuclear pores from nuclear membranes still attached to the lamina.

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Nucleoporins are released from the NE in phase I of NEBD. (A) Maturation of oocytes expressing nucleoporin-GFP fusion proteins coinjected with TRITC 160-kD dextran (only shown for Nup153); arrowhead marks initial entry site. For some nucleoporins a nucleo- or cytoplasmic pool is present in addition to the nuclear rim, and the nucleolus becomes visible (e. g. Nup98). Selected frames are shown, for complete sequences see Videos 4, A–D, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1. Bars, 10 μm. Time, mm:ss. (B) Dissociation kinetics of Nup98 and Nup153. Quantitation of mean fluorescence intensities of the nuclear rim (dashed line in A) of the time series shown in A, normalized from background to maximum values. Vertical black lines mark average time of the start of release determined from independent experiments, gray area indicates standard deviation. (C) Dissociation kinetics of Nup214/CAN and POM121. Quantitation as in B. (D) Thin section electron micrograph of the NE of an immature oocyte. cp, cytoplasm; nu, nucleus; arrowheads, NPCs. Bar, 200 nm. (E) Thin section electron micrograph of the NE of an oocyte at the end of phase I (1–2 min before the initiation of phase II). Labeled as in D. Bar, 200 nm.
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fig5: Nucleoporins are released from the NE in phase I of NEBD. (A) Maturation of oocytes expressing nucleoporin-GFP fusion proteins coinjected with TRITC 160-kD dextran (only shown for Nup153); arrowhead marks initial entry site. For some nucleoporins a nucleo- or cytoplasmic pool is present in addition to the nuclear rim, and the nucleolus becomes visible (e. g. Nup98). Selected frames are shown, for complete sequences see Videos 4, A–D, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1. Bars, 10 μm. Time, mm:ss. (B) Dissociation kinetics of Nup98 and Nup153. Quantitation of mean fluorescence intensities of the nuclear rim (dashed line in A) of the time series shown in A, normalized from background to maximum values. Vertical black lines mark average time of the start of release determined from independent experiments, gray area indicates standard deviation. (C) Dissociation kinetics of Nup214/CAN and POM121. Quantitation as in B. (D) Thin section electron micrograph of the NE of an immature oocyte. cp, cytoplasm; nu, nucleus; arrowheads, NPCs. Bar, 200 nm. (E) Thin section electron micrograph of the NE of an oocyte at the end of phase I (1–2 min before the initiation of phase II). Labeled as in D. Bar, 200 nm.

Mentions: To directly visualize changes in NPC composition we next expressed GFP-tagged mammalian nucleoporins in oocytes by injecting their mRNAs before maturation. The fusion proteins localized correctly to the NE, indicating that they were incorporated into the NPCs (Fig. 5 A). Oocytes expressing chimeric nucleoporins matured with normal kinetics and morphology and imported NLS containing import substrates indicating that the presence of heterologous proteins did not greatly interfere with nucleocytoplasmic transport (unpublished data). We estimated the expression levels of GFP-nucleoporins by comparison with oocytes injected with known amounts of recombinant GFP. For the most highly expressed nucleoporin (Nup153) we calculated ∼2 chimeric proteins per NPC (Fig. S3) while this number was significantly lower for the other nucleoporins.


Nuclear envelope breakdown in starfish oocytes proceeds by partial NPC disassembly followed by a rapidly spreading fenestration of nuclear membranes.

Lénárt P, Rabut G, Daigle N, Hand AR, Terasaki M, Ellenberg J - J. Cell Biol. (2003)

Nucleoporins are released from the NE in phase I of NEBD. (A) Maturation of oocytes expressing nucleoporin-GFP fusion proteins coinjected with TRITC 160-kD dextran (only shown for Nup153); arrowhead marks initial entry site. For some nucleoporins a nucleo- or cytoplasmic pool is present in addition to the nuclear rim, and the nucleolus becomes visible (e. g. Nup98). Selected frames are shown, for complete sequences see Videos 4, A–D, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1. Bars, 10 μm. Time, mm:ss. (B) Dissociation kinetics of Nup98 and Nup153. Quantitation of mean fluorescence intensities of the nuclear rim (dashed line in A) of the time series shown in A, normalized from background to maximum values. Vertical black lines mark average time of the start of release determined from independent experiments, gray area indicates standard deviation. (C) Dissociation kinetics of Nup214/CAN and POM121. Quantitation as in B. (D) Thin section electron micrograph of the NE of an immature oocyte. cp, cytoplasm; nu, nucleus; arrowheads, NPCs. Bar, 200 nm. (E) Thin section electron micrograph of the NE of an oocyte at the end of phase I (1–2 min before the initiation of phase II). Labeled as in D. Bar, 200 nm.
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Related In: Results  -  Collection

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fig5: Nucleoporins are released from the NE in phase I of NEBD. (A) Maturation of oocytes expressing nucleoporin-GFP fusion proteins coinjected with TRITC 160-kD dextran (only shown for Nup153); arrowhead marks initial entry site. For some nucleoporins a nucleo- or cytoplasmic pool is present in addition to the nuclear rim, and the nucleolus becomes visible (e. g. Nup98). Selected frames are shown, for complete sequences see Videos 4, A–D, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1. Bars, 10 μm. Time, mm:ss. (B) Dissociation kinetics of Nup98 and Nup153. Quantitation of mean fluorescence intensities of the nuclear rim (dashed line in A) of the time series shown in A, normalized from background to maximum values. Vertical black lines mark average time of the start of release determined from independent experiments, gray area indicates standard deviation. (C) Dissociation kinetics of Nup214/CAN and POM121. Quantitation as in B. (D) Thin section electron micrograph of the NE of an immature oocyte. cp, cytoplasm; nu, nucleus; arrowheads, NPCs. Bar, 200 nm. (E) Thin section electron micrograph of the NE of an oocyte at the end of phase I (1–2 min before the initiation of phase II). Labeled as in D. Bar, 200 nm.
Mentions: To directly visualize changes in NPC composition we next expressed GFP-tagged mammalian nucleoporins in oocytes by injecting their mRNAs before maturation. The fusion proteins localized correctly to the NE, indicating that they were incorporated into the NPCs (Fig. 5 A). Oocytes expressing chimeric nucleoporins matured with normal kinetics and morphology and imported NLS containing import substrates indicating that the presence of heterologous proteins did not greatly interfere with nucleocytoplasmic transport (unpublished data). We estimated the expression levels of GFP-nucleoporins by comparison with oocytes injected with known amounts of recombinant GFP. For the most highly expressed nucleoporin (Nup153) we calculated ∼2 chimeric proteins per NPC (Fig. S3) while this number was significantly lower for the other nucleoporins.

Bottom Line: In phase II the NE was completely permeabilized within 35 s.This rapid permeabilization spread as a wave from one epicenter on the animal half across the nuclear surface and allowed free diffusion of particles up to approximately 100 nm in diameter into the nucleus.We conclude that NE breakdown in starfish oocytes is triggered by slow sequential disassembly of the NPCs followed by a rapidly spreading fenestration of the NE caused by the removal of nuclear pores from nuclear membranes still attached to the lamina.

View Article: PubMed Central - PubMed

Affiliation: Gene Expression and Cell Biology/Biophysics Programmes, European Molecular Biology Laboratory, D-69117 Heidelberg, Germany.

ABSTRACT
Breakdown of the nuclear envelope (NE) was analyzed in live starfish oocytes using a size series of fluorescently labeled dextrans, membrane dyes, and GFP-tagged proteins of the nuclear pore complex (NPC) and the nuclear lamina. Permeabilization of the nucleus occurred in two sequential phases. In phase I the NE became increasingly permeable for molecules up to approximately 40 nm in diameter, concurrent with a loss of peripheral nuclear pore components over a time course of 10 min. The NE remained intact on the ultrastructural level during this time. In phase II the NE was completely permeabilized within 35 s. This rapid permeabilization spread as a wave from one epicenter on the animal half across the nuclear surface and allowed free diffusion of particles up to approximately 100 nm in diameter into the nucleus. While the lamina and nuclear membranes appeared intact at the light microscopic level, a fenestration of the NE was clearly visible by electron microscopy in phase II. We conclude that NE breakdown in starfish oocytes is triggered by slow sequential disassembly of the NPCs followed by a rapidly spreading fenestration of the NE caused by the removal of nuclear pores from nuclear membranes still attached to the lamina.

Show MeSH
Related in: MedlinePlus