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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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Sequential entry of different size dextrans during phase I of NEBD. (A) Average values and standard deviations of experiments similar to Fig. 2 B. n = 5, 3, 9, 19, and 6 for the 25-, 70-, 90-, 160-, and 500-kD dextrans, respectively. (B) As in A plotted semilogarithmically to compare entry rates and the time differences of the start of entry. A 1% increase was defined as the starting point of entry (see A and Fig. 2 C). (A and B) The <7% of the 160-kD dextran that entered before phase II most likely represent the entry of the smaller molecules in the polydisperse fraction (see Discussion). (C) Entry rates of dextran molecules into the nucleus, calculated for all individual datasets shown in B and averaged (for details see Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1).
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fig3: Sequential entry of different size dextrans during phase I of NEBD. (A) Average values and standard deviations of experiments similar to Fig. 2 B. n = 5, 3, 9, 19, and 6 for the 25-, 70-, 90-, 160-, and 500-kD dextrans, respectively. (B) As in A plotted semilogarithmically to compare entry rates and the time differences of the start of entry. A 1% increase was defined as the starting point of entry (see A and Fig. 2 C). (A and B) The <7% of the 160-kD dextran that entered before phase II most likely represent the entry of the smaller molecules in the polydisperse fraction (see Discussion). (C) Entry rates of dextran molecules into the nucleus, calculated for all individual datasets shown in B and averaged (for details see Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1).

Mentions: To characterize changes in NE permeability during phase I quantitatively, we used dextran fractions of 25–90 kD and compared experiments similar to the one shown in Fig. 2 A from many cells using the start of the 500-kD entry wave to align the series in time. The 25-kD dextran started to enter the nucleus at −11 min and more than 60% of the final nuclear amount had already passed the NE at the end of phase I (Fig. 3, A and B, n = 5). Larger dextran fractions of 70 and 90 kD started later, at −8 and −5.5 min and only 40% and 25% entered in phase I, respectively (Fig. 3, A and B, n = 3 and 9). Although the dextrans started to enter the nucleus at different times, their rates of entry were very similar as is evident from the slopes of the curves in a semilogarithmic plot (Fig. 3 B). These entry kinetics caused by maturation were clearly distinct from the slow and simultaneous entry of all dextran sizes through a single, but relatively large perforation (∼1 μm) produced by a microneedle in the nucleus of an immature oocyte (Fig. S1). Entry kinetics were also not a result of the polydispersity of dextran fractions, because proteins of comparable size (such as MBP; maltose binding protein) showed similar behavior to the dextrans during phase I (Fig. S2).


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)

Sequential entry of different size dextrans during phase I of NEBD. (A) Average values and standard deviations of experiments similar to Fig. 2 B. n = 5, 3, 9, 19, and 6 for the 25-, 70-, 90-, 160-, and 500-kD dextrans, respectively. (B) As in A plotted semilogarithmically to compare entry rates and the time differences of the start of entry. A 1% increase was defined as the starting point of entry (see A and Fig. 2 C). (A and B) The <7% of the 160-kD dextran that entered before phase II most likely represent the entry of the smaller molecules in the polydisperse fraction (see Discussion). (C) Entry rates of dextran molecules into the nucleus, calculated for all individual datasets shown in B and averaged (for details see Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1).
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Related In: Results  -  Collection

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fig3: Sequential entry of different size dextrans during phase I of NEBD. (A) Average values and standard deviations of experiments similar to Fig. 2 B. n = 5, 3, 9, 19, and 6 for the 25-, 70-, 90-, 160-, and 500-kD dextrans, respectively. (B) As in A plotted semilogarithmically to compare entry rates and the time differences of the start of entry. A 1% increase was defined as the starting point of entry (see A and Fig. 2 C). (A and B) The <7% of the 160-kD dextran that entered before phase II most likely represent the entry of the smaller molecules in the polydisperse fraction (see Discussion). (C) Entry rates of dextran molecules into the nucleus, calculated for all individual datasets shown in B and averaged (for details see Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1).
Mentions: To characterize changes in NE permeability during phase I quantitatively, we used dextran fractions of 25–90 kD and compared experiments similar to the one shown in Fig. 2 A from many cells using the start of the 500-kD entry wave to align the series in time. The 25-kD dextran started to enter the nucleus at −11 min and more than 60% of the final nuclear amount had already passed the NE at the end of phase I (Fig. 3, A and B, n = 5). Larger dextran fractions of 70 and 90 kD started later, at −8 and −5.5 min and only 40% and 25% entered in phase I, respectively (Fig. 3, A and B, n = 3 and 9). Although the dextrans started to enter the nucleus at different times, their rates of entry were very similar as is evident from the slopes of the curves in a semilogarithmic plot (Fig. 3 B). These entry kinetics caused by maturation were clearly distinct from the slow and simultaneous entry of all dextran sizes through a single, but relatively large perforation (∼1 μm) produced by a microneedle in the nucleus of an immature oocyte (Fig. S1). Entry kinetics were also not a result of the polydispersity of dextran fractions, because proteins of comparable size (such as MBP; maltose binding protein) showed similar behavior to the dextrans during phase I (Fig. S2).

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