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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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Import substrates are released from the nucleus during phase I of NEBD. (A) Oocytes coinjected with Alexa 594 IBB-MBP or M9-MBP and Alexa 488 MBP into the nucleus and Cy5 500-kD dextran into the cytoplasm (MBP and dextran shown only for M9-MBP). Selected frames are shown, for complete sequences see Video 3, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1. Bar, 25 μm. Time, mm:ss. Arrowhead, the dextran entry wave. (B) Quantitation of mean nuclear (dashed lines in A) fluorescence intensities of the time series shown in A, normalized from minimum to maximum values. The two sequences were aligned by the 500-kD dextran entry. The slow entry of the 500-kD dextran before 0 min is due to the small hole created by the nuclear injection.
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fig4: Import substrates are released from the nucleus during phase I of NEBD. (A) Oocytes coinjected with Alexa 594 IBB-MBP or M9-MBP and Alexa 488 MBP into the nucleus and Cy5 500-kD dextran into the cytoplasm (MBP and dextran shown only for M9-MBP). Selected frames are shown, for complete sequences see Video 3, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1. Bar, 25 μm. Time, mm:ss. Arrowhead, the dextran entry wave. (B) Quantitation of mean nuclear (dashed lines in A) fluorescence intensities of the time series shown in A, normalized from minimum to maximum values. The two sequences were aligned by the 500-kD dextran entry. The slow entry of the 500-kD dextran before 0 min is due to the small hole created by the nuclear injection.

Mentions: If the apparent diameter of the NPC diffusion pore increases from 12 to 40 nm during phase I of NEBD due to NPC disassembly, we would predict defects in nucleocytoplasmic transport at this time. To test this prediction, we used well-characterized import substrates, MBP fused to an importin-β binding domain (IBB-MBP) and a transportin specific import signal (M9-MBP), both labeled with Alexa 594 (Ribbeck and Görlich, 2002; Katharina Ribbeck, personal communication). Both proteins were efficiently imported into the nucleus of starfish oocytes (unpublished data). To simultaneously follow the changes in active transport and passive diffusion we coinjected M9- or IBB-MBP with MBP (without import signal) into the nucleus of oocytes together with a cytoplasmically injected large dextran (500 kD) (Fig. 4, Videos 3, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1). M9-MBP was released with kinetics strikingly similar to MBP (Fig. 4, A and B) starting at −6.8 ± 1 min (n = 11). At the end of phase I, >70% of the proteins had been released into the cytoplasm. The release of IBB-MBP started slightly later (−3.8 ± 0.6 min, n = 6) and showed a different kinetics, which might be a result of the retention of IBB-MBP by binding to other nuclear factors (Fig. 4, A and B). The release of two different types of import substrates from the nucleus during phase I further suggested that NPC disassembly is responsible for the increased NE permeability in phase I and also indicates the dual role of the NPC controlling nuclear transport and passive diffusion through the NE.


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)

Import substrates are released from the nucleus during phase I of NEBD. (A) Oocytes coinjected with Alexa 594 IBB-MBP or M9-MBP and Alexa 488 MBP into the nucleus and Cy5 500-kD dextran into the cytoplasm (MBP and dextran shown only for M9-MBP). Selected frames are shown, for complete sequences see Video 3, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1. Bar, 25 μm. Time, mm:ss. Arrowhead, the dextran entry wave. (B) Quantitation of mean nuclear (dashed lines in A) fluorescence intensities of the time series shown in A, normalized from minimum to maximum values. The two sequences were aligned by the 500-kD dextran entry. The slow entry of the 500-kD dextran before 0 min is due to the small hole created by the nuclear injection.
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Related In: Results  -  Collection

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fig4: Import substrates are released from the nucleus during phase I of NEBD. (A) Oocytes coinjected with Alexa 594 IBB-MBP or M9-MBP and Alexa 488 MBP into the nucleus and Cy5 500-kD dextran into the cytoplasm (MBP and dextran shown only for M9-MBP). Selected frames are shown, for complete sequences see Video 3, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1. Bar, 25 μm. Time, mm:ss. Arrowhead, the dextran entry wave. (B) Quantitation of mean nuclear (dashed lines in A) fluorescence intensities of the time series shown in A, normalized from minimum to maximum values. The two sequences were aligned by the 500-kD dextran entry. The slow entry of the 500-kD dextran before 0 min is due to the small hole created by the nuclear injection.
Mentions: If the apparent diameter of the NPC diffusion pore increases from 12 to 40 nm during phase I of NEBD due to NPC disassembly, we would predict defects in nucleocytoplasmic transport at this time. To test this prediction, we used well-characterized import substrates, MBP fused to an importin-β binding domain (IBB-MBP) and a transportin specific import signal (M9-MBP), both labeled with Alexa 594 (Ribbeck and Görlich, 2002; Katharina Ribbeck, personal communication). Both proteins were efficiently imported into the nucleus of starfish oocytes (unpublished data). To simultaneously follow the changes in active transport and passive diffusion we coinjected M9- or IBB-MBP with MBP (without import signal) into the nucleus of oocytes together with a cytoplasmically injected large dextran (500 kD) (Fig. 4, Videos 3, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1). M9-MBP was released with kinetics strikingly similar to MBP (Fig. 4, A and B) starting at −6.8 ± 1 min (n = 11). At the end of phase I, >70% of the proteins had been released into the cytoplasm. The release of IBB-MBP started slightly later (−3.8 ± 0.6 min, n = 6) and showed a different kinetics, which might be a result of the retention of IBB-MBP by binding to other nuclear factors (Fig. 4, A and B). The release of two different types of import substrates from the nucleus during phase I further suggested that NPC disassembly is responsible for the increased NE permeability in phase I and also indicates the dual role of the NPC controlling nuclear transport and passive diffusion through the NE.

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