Lénárt P et al. (2003), Nuclear envelope breakdown in starfish oocytes ...

ECB-ART-38025

J Cell Biol 2003 Mar 31;1607:1055-68. doi: 10.1083/jcb.200211076.

Show Gene links Show Anatomy links

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 .

???displayArticle.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.

???displayArticle.pubmedLink??? 12654902
???displayArticle.pmcLink??? PMC2172766
???displayArticle.link??? J Cell Biol

Species referenced: Echinodermata
Genes referenced: LOC100893907 LOC115918707 LOC576396 LOC590120 nup153 nup98 pole

???attribute.lit??? ???displayArticles.show???

	Figure 1. NE permeability in immature oocytes. (A) Immature starfish oocyte injected with Alexa 488 10-kD dextran and imaged immediately after injection. Dextran diffuses from the vegetal pole through the cytoplasm and simultaneously fills the nucleus. Note accumulation of dextran adjacent to the nuclear rim at early times, indicating that nuclear entry is slower than diffusion. Selected frames are shown, for complete sequence see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1. Bar, 25 Î¼m. Time, mm:ss. (B) Time series of an immature starfish oocyte injected with Alexa 488 25-kD dextran. Scale and time as in A. (C) Quantitation of mean nuclear (dashed lines in A and B) fluorescence over time of the sequences shown in A and B. Intensities were normalized from initial to final values (A), or from minimum to two times the average cytoplasmic values (B). (D) High-resolution confocal image of a 10-kD dextran-injected oocyte after equilibration. Cytoplasm is filled with yolk platelets, resulting in an apparent 50% lower concentration. High magnification inset (bottom) shows single platelets and a yolk-free zone adjacent to the NE (arrowheads). Bars: 10 (top) and 2 Î¼m (bottom).
	Figure 2. 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 Î¼m. 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.
	Figure 3. 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).
	Figure 4. 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 Î¼m. 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.
	Figure 5. Nucleoporins are released from the NE in phase I of NEBD. (A) Maturation of oocytes expressing nucleoporin-GFP fusion proteins coinjected with TRITC 160-kD dextran (only shown for Nup153); arrowhead marks initial entry site. For some nucleoporins a nucleo- or cytoplasmic pool is present in addition to the nuclear rim, and the nucleolus becomes visible (e. g. Nup98). Selected frames are shown, for complete sequences see Videos 4, Aâ€“D, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1. Bars, 10 Î¼m. Time, mm:ss. (B) Dissociation kinetics of Nup98 and Nup153. Quantitation of mean fluorescence intensities of the nuclear rim (dashed line in A) of the time series shown in A, normalized from background to maximum values. Vertical black lines mark average time of the start of release determined from independent experiments, gray area indicates standard deviation. (C) Dissociation kinetics of Nup214/CAN and POM121. Quantitation as in B. (D) Thin section electron micrograph of the NE of an immature oocyte. cp, cytoplasm; nu, nucleus; arrowheads, NPCs. Bar, 200 nm. (E) Thin section electron micrograph of the NE of an oocyte at the end of phase I (1â€“2 min before the initiation of phase II). Labeled as in D. Bar, 200 nm.
	Figure 6. 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 Î¼m. (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.
	Figure 7. 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 Î¼m. 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 Î¼m (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 Î¼m.
	Figure 8. Model for phase I and II of NEBD. (Top) cross section of the NE through the center of an NPC. (Bottom) top view from the cytoplasmic side of the NE. Lamina, dark gray; nuclear membranes, transparent gray; NPC, middle gray; permeability barrier of NPC, light gray. The channel diameter indicated by dashed lines corresponds to the apparent diffusion channel calculated from dextran fluxes. (A) immature oocytes, (B) phase I oocytes, (C) phase II oocytes. Bar, 50 nm.

References [+] :

Akey, Architecture of the Xenopus nuclear pore complex revealed by three-dimensional cryo-electron microscopy. 1993, Pubmed