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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 μm. 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 μm (bottom).
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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 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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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).
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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 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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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, A–D, available at http://www.jcb.org/cgi/content/full/jcb.200211076/DC1. Bars, 10 μm. 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 (1–2 min before the initiation of phase II). Labeled as in D. Bar, 200 nm.
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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 μm. (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.
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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 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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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.
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