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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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Fenestration of the NE in phase II. The lamina and nuclear membranes remain intact on the light microscopic level. (A) Oocyte expressing Lamin B1-GFP coinjected with the lipophilic dye DiIC16 and Cy5 500-kD dextran. Selected frames are shown, for complete sequence see Video 6, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1. Bar, 10 μm. Time, mm:ss, 0:00 = start of 500-kD entry. Arrowhead marks first visible gap. (B) Thin section electron micrograph of the NE of an oocyte at the initiation of phase II. cp, cytoplasm; nu, nucleus; arrowheads, disassembling NPCs; arrows, gaps on the NE. Dashed rectangles mark regions shown in the bottom panels at higher magnification. Bar, 5 μm (top) and 200 nm (bottom). (C) Thin section electron micrograph of the NE of an oocyte during phase II, at a later stage than B. Labeled as in B. Bar, 1 μm.
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fig7: Fenestration of the NE in phase II. The lamina and nuclear membranes remain intact on the light microscopic level. (A) Oocyte expressing Lamin B1-GFP coinjected with the lipophilic dye DiIC16 and Cy5 500-kD dextran. Selected frames are shown, for complete sequence see Video 6, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1. Bar, 10 μm. Time, mm:ss, 0:00 = start of 500-kD entry. Arrowhead marks first visible gap. (B) Thin section electron micrograph of the NE of an oocyte at the initiation of phase II. cp, cytoplasm; nu, nucleus; arrowheads, disassembling NPCs; arrows, gaps on the NE. Dashed rectangles mark regions shown in the bottom panels at higher magnification. Bar, 5 μm (top) and 200 nm (bottom). (C) Thin section electron micrograph of the NE of an oocyte during phase II, at a later stage than B. Labeled as in B. Bar, 1 μm.

Mentions: Since peripheral nucleoporins were almost completely lost from the NE during the time in which NPC permeabilization spread over the NE (Fig. 5 A), we investigated if other NE structures such as the lamina and the nuclear membranes were also disassembled at this time. Indication that parts of the NE structure must remain intact during phase II came from the observation that a clear boundary between the cytoplasm and nucleus was observed during phase I and II of NEBD by DIC (Fig. 2 A), 500-kD dextran (Fig. 6 A), and POM121 fluorescence (Fig. 5 A). Although this boundary was completely permeable for molecules up to 93-nm diameter, it still prevented the entry of larger particles such as yolk platelets into the nucleoplasm (Fig. 7 A, DIC). Nuclear membranes and the lamina were labeled with the membrane dye DiIC16 and GFP tagged lamin B, respectively, and visualized directly during NEBD together with a 500-kD dextran in the same cell (Fig. 7 A, see also Video 6, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1). Such time-lapse sequences (n = 5) showed that the dextran wave entered through the lamina and nuclear membranes that appeared intact by confocal microscopy and could thus not contain gaps >1 μm (Fig. 7 A, 0:00). Indeed, on electron micrographs of oocytes fixed at the beginning of phase II, gaps with a size of 100–200 nm could be seen (Fig. 7, B and C), which would account for the rapid diffusion of the largest dextran molecules, but still escape the resolution of the confocal microscope. At this stage the NE consisted of membrane elements of 1–2 μm in length containing 3–10 NPC-like structures, which appeared to be in different stages of disassembly (Fig. 7, B and C). This is consistent with our light microscopy data that all peripheral nucleoporins are released at this time while a significant fraction of POM121 fluorescence remained associated with the NE (Fig. 5, A and C). By light microscopy large discontinuities could only be detected several minutes later. These holes appeared simultaneously in the polymerized lamina and nuclear membranes that were still attached to the lamina (Fig. 7 A). The lamina was completely solubilized >10 min after the beginning of phase II (unpublished data).


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)

Fenestration of the NE in phase II. The lamina and nuclear membranes remain intact on the light microscopic level. (A) Oocyte expressing Lamin B1-GFP coinjected with the lipophilic dye DiIC16 and Cy5 500-kD dextran. Selected frames are shown, for complete sequence see Video 6, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1. Bar, 10 μm. Time, mm:ss, 0:00 = start of 500-kD entry. Arrowhead marks first visible gap. (B) Thin section electron micrograph of the NE of an oocyte at the initiation of phase II. cp, cytoplasm; nu, nucleus; arrowheads, disassembling NPCs; arrows, gaps on the NE. Dashed rectangles mark regions shown in the bottom panels at higher magnification. Bar, 5 μm (top) and 200 nm (bottom). (C) Thin section electron micrograph of the NE of an oocyte during phase II, at a later stage than B. Labeled as in B. Bar, 1 μm.
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fig7: Fenestration of the NE in phase II. The lamina and nuclear membranes remain intact on the light microscopic level. (A) Oocyte expressing Lamin B1-GFP coinjected with the lipophilic dye DiIC16 and Cy5 500-kD dextran. Selected frames are shown, for complete sequence see Video 6, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1. Bar, 10 μm. Time, mm:ss, 0:00 = start of 500-kD entry. Arrowhead marks first visible gap. (B) Thin section electron micrograph of the NE of an oocyte at the initiation of phase II. cp, cytoplasm; nu, nucleus; arrowheads, disassembling NPCs; arrows, gaps on the NE. Dashed rectangles mark regions shown in the bottom panels at higher magnification. Bar, 5 μm (top) and 200 nm (bottom). (C) Thin section electron micrograph of the NE of an oocyte during phase II, at a later stage than B. Labeled as in B. Bar, 1 μm.
Mentions: Since peripheral nucleoporins were almost completely lost from the NE during the time in which NPC permeabilization spread over the NE (Fig. 5 A), we investigated if other NE structures such as the lamina and the nuclear membranes were also disassembled at this time. Indication that parts of the NE structure must remain intact during phase II came from the observation that a clear boundary between the cytoplasm and nucleus was observed during phase I and II of NEBD by DIC (Fig. 2 A), 500-kD dextran (Fig. 6 A), and POM121 fluorescence (Fig. 5 A). Although this boundary was completely permeable for molecules up to 93-nm diameter, it still prevented the entry of larger particles such as yolk platelets into the nucleoplasm (Fig. 7 A, DIC). Nuclear membranes and the lamina were labeled with the membrane dye DiIC16 and GFP tagged lamin B, respectively, and visualized directly during NEBD together with a 500-kD dextran in the same cell (Fig. 7 A, see also Video 6, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1). Such time-lapse sequences (n = 5) showed that the dextran wave entered through the lamina and nuclear membranes that appeared intact by confocal microscopy and could thus not contain gaps >1 μm (Fig. 7 A, 0:00). Indeed, on electron micrographs of oocytes fixed at the beginning of phase II, gaps with a size of 100–200 nm could be seen (Fig. 7, B and C), which would account for the rapid diffusion of the largest dextran molecules, but still escape the resolution of the confocal microscope. At this stage the NE consisted of membrane elements of 1–2 μm in length containing 3–10 NPC-like structures, which appeared to be in different stages of disassembly (Fig. 7, B and C). This is consistent with our light microscopy data that all peripheral nucleoporins are released at this time while a significant fraction of POM121 fluorescence remained associated with the NE (Fig. 5, A and C). By light microscopy large discontinuities could only be detected several minutes later. These holes appeared simultaneously in the polymerized lamina and nuclear membranes that were still attached to the lamina (Fig. 7 A). The lamina was completely solubilized >10 min after the beginning of phase II (unpublished data).

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