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Science 21 April 2006:
Vol. 312. no. 5772, pp. 440 - 443
DOI: 10.1126/science.1124196
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Reports
Nuclear Pores Form de Novo from Both Sides of the Nuclear Envelope
Maximiliano A. D'Angelo*,
Daniel J. Anderson*,
Erin Richard and
Martin W. Hetzer
Salk Institute for Biological Studies, Molecular and Cell Biology Laboratory, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.
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Fig. 1. Transport-independent role of Importin ß in NPC insertion. (A) Nuclei (NE-0) were preassembled in 5-µl reactions including egg extracts, membranes, and sperm chromatin for 40 min (17), and diluted with 50 µl fresh cytosol in the absence or presence of 5 µM RanT24N, 2 mM GTP S, WGA (20 µg/ml), 2 µM transportin (Trn), or Importin ß (Imp ß) (±10 µM RanGTP), for an additional 80 min (NE-80) and analyzed by confocal microscopy. The formation of a closed NE was analyzed in unfixed nuclei using the membrane stain DiIC16 (1,1'dihexadecyl-3,3,3',3'-tetramethylin-docarbocyanine perchlorate) (27) and 70-kD dextran (small insert). Individual NPCs were visualized by immunofluorescence with mAb414 on the surface of the nuclei at low magnification (middle row) and with 8x zoom (bottom row). Scale bars, 10 µm. (B) NE surface area and total number of pores were calculated using five independent experiments as described in (28). The relative increase of surface area (black) and pore numbers (gray) during 80 min were plotted. Error bars indicate SD. (C) Nuclei were assembled in the absence (buffer) and presence of 2 µM Importin ß, sectioned, and analyzed by transmission electron microscopy (17). Scale bar, 200 nm. Cyto, cytosol.
[View Larger Version of this Image (102K GIF file)]
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Fig. 2. RanGTP-mediated incorporation of Nup107-160 complex occurs from the nucleoplasmic and cytoplasmic side of the NE. (A) Fluorescently labeled RanT24N accumulated in assembled nuclei and was trapped in the nucleoplasm of WGA-sealed nuclei after dilution (T24N  WGA). RanT24N was excluded from the nucleoplasm when added after the NPCs were blocked with WGA (WGA T24N). Scale bar, 10 µm. (B) The area labeled "control" shows the quantification of total pore numbers of NE-0 and NE-80 nuclei. The area labeled "T24N" shows data for NE-0 nuclei that were incubated with 5 µM RanT24N for 10 min. NPC insertion was induced with 200 volumes of cytosol (cyto) or 5 µM RCC1. To trap RanT24N inside the nucleus, NPCs were sealed with WGA (20 µg/ml) for 10 min, and NPC insertion was then induced by adding cytosol (WGA cyto) or 5 µM RanGTP (WGA RanQ69L). The area labeled "WGA" shows data for NE-0 nuclei that were incubated with WGA (20 µg/ml) for 10 min before the addition of cytosol (cyto), cytosol that contained 5 µM RanT24N (cyto[T24N]), or RanT24N and RanQ69L (cyto[T24N] Q69L). The number of NPCs was quantified as described in Fig. 1. (C) NE-0 nuclei were incubated in the absence or presence of 5 µM RanGAP-RanBP1 or RanGAP-RanBP1 plus 5 µM RCC1 as indicated. Nuclei were stained with mAb414 (green) and DiIC16 (red) and analyzed in cross sections or (D) on the NE surface to visualize individual NPCs. Scale bars, 10 µm.
[View Larger Version of this Image (58K GIF file)]
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Fig. 3. Nucleoplasmic and cytoplasmic Nup107-160 complexes are required for NPC insertion. (A) NE-0 nuclei were incubated in 5 µl fresh cytosol to which buffer or 2 µM Importin ß had been added, and nuclei were analyzed after 80 min by immunofluorescence using  Nup133 and mAb414. To reverse the inhibitory effect of Importin ß, NE-80 nuclei were diluted in 200 volumes of cytosol. Scale bar, 10 µm. (B) Nuclei were assembled in the presence of Importin ß, and subsequently diluted in 200 volumes of cytosol (buffer), cytosol containing 5 µM RanT24N, or 10 µM RanGAP-RanBP1 for 30 min. Nuclei were analyzed by immunofluorescence using Nup133. Scale bar, 10 µm. (C) NE-0 nuclei were incubated at room temperature with mock-depleted or Nup107-160–depleted cytosol (in the absence or presence of purified Nup107-160 complex), and the total number of NPCs was quantified as in Fig. 1. Error bars indicate SD. (D) Nuclear assembly was performed using DiIC16-labeled NE membranes, sperm chromatin, and cytosol in the presence of 5 mM BAPTA for 60 min, and the nuclei were diluted with mock-depleted cytosol or Nup107-160–depleted cytosol. NPCs were visualized with mAb414 and Nup133. Scale bar, 10 µm.
[View Larger Version of this Image (80K GIF file)]
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Fig. 4. NPC insertion occurs by a de novo mechanism. (A) NE-0 nuclei were labeled with fluorescently labeled WGA-488 (10 µg/ml) for 10 min and subsequently incubated with 400 volumes of cytosol in the absence or presence of 5 µM RanT24N for 30 min. Fluorescently labeled WGA-568 was added for 10 min, and nuclei were analyzed by confocal microscopy. Newly formed pores are visible as red dots; they do not contain a detectable green signal. Scale bar, 1 µm. (B) HeLa cells expressing POM121-(GFP3) were analyzed in real time by confocal microscopy on the NE surface every 2 min. Arrows indicate new NPC assembly site. (C) Relative fluorescence intensity of preexisting pores (gray) and new pores (red) was plotted against time. (D) Large areas of NEs were photobleached (inset), and the formation of few pores was monitored by 4D confocal microscopy. Bleached POM121-(GFP3) did not recover for more than 4 hours, similar to previously published report (29).
[View Larger Version of this Image (80K GIF file)]
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