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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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Two phases of dextran entry during NEBD. (A) Oocyte coinjected with 500, 90, and 70 kD dextrans labeled with Alexa 488, Cy5, and TMR, respectively. Arrowheads mark the start of complete permeabilization also visible by DIC. At 2:00 the 500-kD dextran is equilibrated only to 90% due to its slow diffusion. Small dark circle in the cytoplasm is an oil drop from the injection. Fluorescence images are pseudo colored for easier comparison. Selected frames are shown, for complete sequence see Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1. Bar, 25 μm. Time mm:ss, 0:00 = start of 500-kD dextran entry. (B) Quantitation of mean nuclear (dashed lines in A) fluorescence of time series shown in A, normalized from minimum to maximum values. Arrows mark time points shown in A. (C) As in B, plotted semilogarithmically to compare entry rates.
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fig2: Two phases of dextran entry during NEBD. (A) Oocyte coinjected with 500, 90, and 70 kD dextrans labeled with Alexa 488, Cy5, and TMR, respectively. Arrowheads mark the start of complete permeabilization also visible by DIC. At 2:00 the 500-kD dextran is equilibrated only to 90% due to its slow diffusion. Small dark circle in the cytoplasm is an oil drop from the injection. Fluorescence images are pseudo colored for easier comparison. Selected frames are shown, for complete sequence see Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1. Bar, 25 μm. Time mm:ss, 0:00 = start of 500-kD dextran entry. (B) Quantitation of mean nuclear (dashed lines in A) fluorescence of time series shown in A, normalized from minimum to maximum values. Arrows mark time points shown in A. (C) As in B, plotted semilogarithmically to compare entry rates.

Mentions: To measure the permeability of the intact NE in immature oocytes, we injected different sizes of fluorescently labeled dextrans (Table I; see also Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1) into the cytoplasm and monitored their ability to enter the nucleus by measuring the increase of mean fluorescence intensity in the nuclear region over time. A 10-kD dextran entered the nucleus of immature oocytes rapidly by passive diffusion and equilibrated between nucleus and cytoplasm in ∼30 min (Fig. 1, A and C). The two times higher fluorescence intensity in the nucleus as compared with the cytoplasm after equilibration is due to yolk platelets that occupy ∼50% of the cytoplasmic volume, except for a small rim of yolk-free cytosol directly adjacent to the NE (Fig. 1 D). Dextrans with larger molecular weights (MW), such as 25 kD, were excluded during this time (Fig. 1, B and C), and even after 2 wk <50% had entered the nucleus. Fractions above 25 kD did not enter the nucleus at all even during such prolonged periods (unpublished data). Thus, in immature oocytes, dextrans with diameters of ∼10 nm (10 kD) were able to pass through intact nuclear pores, whereas NPCs efficiently excluded larger molecules from the nucleus. Dextrans of 25 kD or larger (≥20 nm in diameter) were only able to enter the nucleoplasm during NEBD, at oocyte maturation (Terasaki, 1994; Fig. 2 A).


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)

Two phases of dextran entry during NEBD. (A) Oocyte coinjected with 500, 90, and 70 kD dextrans labeled with Alexa 488, Cy5, and TMR, respectively. Arrowheads mark the start of complete permeabilization also visible by DIC. At 2:00 the 500-kD dextran is equilibrated only to 90% due to its slow diffusion. Small dark circle in the cytoplasm is an oil drop from the injection. Fluorescence images are pseudo colored for easier comparison. Selected frames are shown, for complete sequence see Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1. Bar, 25 μm. Time mm:ss, 0:00 = start of 500-kD dextran entry. (B) Quantitation of mean nuclear (dashed lines in A) fluorescence of time series shown in A, normalized from minimum to maximum values. Arrows mark time points shown in A. (C) As in B, plotted semilogarithmically to compare entry rates.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172766&req=5

fig2: Two phases of dextran entry during NEBD. (A) Oocyte coinjected with 500, 90, and 70 kD dextrans labeled with Alexa 488, Cy5, and TMR, respectively. Arrowheads mark the start of complete permeabilization also visible by DIC. At 2:00 the 500-kD dextran is equilibrated only to 90% due to its slow diffusion. Small dark circle in the cytoplasm is an oil drop from the injection. Fluorescence images are pseudo colored for easier comparison. Selected frames are shown, for complete sequence see Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1. Bar, 25 μm. Time mm:ss, 0:00 = start of 500-kD dextran entry. (B) Quantitation of mean nuclear (dashed lines in A) fluorescence of time series shown in A, normalized from minimum to maximum values. Arrows mark time points shown in A. (C) As in B, plotted semilogarithmically to compare entry rates.
Mentions: To measure the permeability of the intact NE in immature oocytes, we injected different sizes of fluorescently labeled dextrans (Table I; see also Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1) into the cytoplasm and monitored their ability to enter the nucleus by measuring the increase of mean fluorescence intensity in the nuclear region over time. A 10-kD dextran entered the nucleus of immature oocytes rapidly by passive diffusion and equilibrated between nucleus and cytoplasm in ∼30 min (Fig. 1, A and C). The two times higher fluorescence intensity in the nucleus as compared with the cytoplasm after equilibration is due to yolk platelets that occupy ∼50% of the cytoplasmic volume, except for a small rim of yolk-free cytosol directly adjacent to the NE (Fig. 1 D). Dextrans with larger molecular weights (MW), such as 25 kD, were excluded during this time (Fig. 1, B and C), and even after 2 wk <50% had entered the nucleus. Fractions above 25 kD did not enter the nucleus at all even during such prolonged periods (unpublished data). Thus, in immature oocytes, dextrans with diameters of ∼10 nm (10 kD) were able to pass through intact nuclear pores, whereas NPCs efficiently excluded larger molecules from the nucleus. Dextrans of 25 kD or larger (≥20 nm in diameter) were only able to enter the nucleoplasm during NEBD, at oocyte maturation (Terasaki, 1994; Fig. 2 A).

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