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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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Permeabilization wave of the NE during phase II of NEBD. Oocyte injected with Alexa 488 500-kD dextran imaged in 3D over time during maturation. See also Video 5, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1. Bars, 10 μm. (A) Selected optical slice from the 4D dataset showing initiation and spreading of the permeabilization. Arrowheads mark the boundary between permeabilized and intact NE. The nucleolus excludes the dextran and becomes visible in the last two frames. (B) Isosurface visualization of the complete 4D dataset. Intact (light gray) and permeabilized (dark gray) areas of the NE were segmented and reconstructed in 3D. (C) Animal-vegetal optical cross section of the 4D dataset shown in A and B. Permeabilization is initiated between the animal pole and equator of the nucleus (arrowheads). (D) Positions of the initial entry site from 10 experiments analyzed as in C. Distance from the animal pole normalized to the height of the nucleus is plotted on a scheme of the nuclear surface. (E) Line profiles of fluorescence intensity along the primary entry site (shown by a dashed rectangle on A) at different time points. cp, cytoplasm; nu, nucleus.
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fig6: Permeabilization wave of the NE during phase II of NEBD. Oocyte injected with Alexa 488 500-kD dextran imaged in 3D over time during maturation. See also Video 5, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1. Bars, 10 μm. (A) Selected optical slice from the 4D dataset showing initiation and spreading of the permeabilization. Arrowheads mark the boundary between permeabilized and intact NE. The nucleolus excludes the dextran and becomes visible in the last two frames. (B) Isosurface visualization of the complete 4D dataset. Intact (light gray) and permeabilized (dark gray) areas of the NE were segmented and reconstructed in 3D. (C) Animal-vegetal optical cross section of the 4D dataset shown in A and B. Permeabilization is initiated between the animal pole and equator of the nucleus (arrowheads). (D) Positions of the initial entry site from 10 experiments analyzed as in C. Distance from the animal pole normalized to the height of the nucleus is plotted on a scheme of the nuclear surface. (E) Line profiles of fluorescence intensity along the primary entry site (shown by a dashed rectangle on A) at different time points. cp, cytoplasm; nu, nucleus.

Mentions: Phase II of NEBD was marked by an entry wave of even very large macromolecules into the nucleus. This wave initiated at a single location on the NE and then rapidly spread across the nuclear surface (Fig. 6 A, see also Video 5 A). The large 500-kD dextran with 93-nm diameter could freely enter the nucleus through the permeabilized regions of the NE at this time (Fig. 6, A and E). The local fluxes measured at the permeabilized areas were about five times higher than the peak flux at the end of the first phase (see Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1) and the kinetics of phase II also fitted well to simulating free diffusion of dextran through an expanding hole into the nucleus (Terasaki et al., 2001). In this way the nucleus equilibrated with the cytoplasmic dextrans in <1 min.


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)

Permeabilization wave of the NE during phase II of NEBD. Oocyte injected with Alexa 488 500-kD dextran imaged in 3D over time during maturation. See also Video 5, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1. Bars, 10 μm. (A) Selected optical slice from the 4D dataset showing initiation and spreading of the permeabilization. Arrowheads mark the boundary between permeabilized and intact NE. The nucleolus excludes the dextran and becomes visible in the last two frames. (B) Isosurface visualization of the complete 4D dataset. Intact (light gray) and permeabilized (dark gray) areas of the NE were segmented and reconstructed in 3D. (C) Animal-vegetal optical cross section of the 4D dataset shown in A and B. Permeabilization is initiated between the animal pole and equator of the nucleus (arrowheads). (D) Positions of the initial entry site from 10 experiments analyzed as in C. Distance from the animal pole normalized to the height of the nucleus is plotted on a scheme of the nuclear surface. (E) Line profiles of fluorescence intensity along the primary entry site (shown by a dashed rectangle on A) at different time points. cp, cytoplasm; nu, nucleus.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172766&req=5

fig6: Permeabilization wave of the NE during phase II of NEBD. Oocyte injected with Alexa 488 500-kD dextran imaged in 3D over time during maturation. See also Video 5, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1. Bars, 10 μm. (A) Selected optical slice from the 4D dataset showing initiation and spreading of the permeabilization. Arrowheads mark the boundary between permeabilized and intact NE. The nucleolus excludes the dextran and becomes visible in the last two frames. (B) Isosurface visualization of the complete 4D dataset. Intact (light gray) and permeabilized (dark gray) areas of the NE were segmented and reconstructed in 3D. (C) Animal-vegetal optical cross section of the 4D dataset shown in A and B. Permeabilization is initiated between the animal pole and equator of the nucleus (arrowheads). (D) Positions of the initial entry site from 10 experiments analyzed as in C. Distance from the animal pole normalized to the height of the nucleus is plotted on a scheme of the nuclear surface. (E) Line profiles of fluorescence intensity along the primary entry site (shown by a dashed rectangle on A) at different time points. cp, cytoplasm; nu, nucleus.
Mentions: Phase II of NEBD was marked by an entry wave of even very large macromolecules into the nucleus. This wave initiated at a single location on the NE and then rapidly spread across the nuclear surface (Fig. 6 A, see also Video 5 A). The large 500-kD dextran with 93-nm diameter could freely enter the nucleus through the permeabilized regions of the NE at this time (Fig. 6, A and E). The local fluxes measured at the permeabilized areas were about five times higher than the peak flux at the end of the first phase (see Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1) and the kinetics of phase II also fitted well to simulating free diffusion of dextran through an expanding hole into the nucleus (Terasaki et al., 2001). In this way the nucleus equilibrated with the cytoplasmic dextrans in <1 min.

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