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Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells.

Daigle N, Beaudouin J, Hartnell L, Imreh G, Hallberg E, Lippincott-Schwartz J, Ellenberg J - J. Cell Biol. (2001)

Bottom Line: No independent movement of single pore complexes was found within the plane of the NE in interphase.During mitosis, POM121 and Nup153 were completely dispersed and mobile in the ER (POM121) or cytosol (Nup153) in metaphase, and rapidly redistributed to an immobilized pool around chromatin in late anaphase.Assembly and immobilization of both nucleoporins occurred before detectable recruitment of lamin B1, which is thus unlikely to mediate initiation of NPC assembly at the end of mitosis.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.

ABSTRACT
The nuclear pore complex (NPC) and its relationship to the nuclear envelope (NE) was characterized in living cells using POM121-green fluorescent protein (GFP) and GFP-Nup153, and GFP-lamin B1. No independent movement of single pore complexes was found within the plane of the NE in interphase. Only large arrays of NPCs moved slowly and synchronously during global changes in nuclear shape, strongly suggesting mechanical connections which form an NPC network. The nuclear lamina exhibited identical movements. NPC turnover measured by fluorescence recovery after photobleaching of POM121 was less than once per cell cycle. Nup153 association with NPCs was dynamic and turnover of this nucleoporin was three orders of magnitude faster. Overexpression of both nucleoporins induced the formation of annulate lamellae (AL) in the endoplasmic reticulum (ER). Turnover of AL pore complexes was much higher than in the NE (once every 2.5 min). During mitosis, POM121 and Nup153 were completely dispersed and mobile in the ER (POM121) or cytosol (Nup153) in metaphase, and rapidly redistributed to an immobilized pool around chromatin in late anaphase. Assembly and immobilization of both nucleoporins occurred before detectable recruitment of lamin B1, which is thus unlikely to mediate initiation of NPC assembly at the end of mitosis.

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POM121-GFP and GFP-Nup153 label single NPCs and induce AL in live cells. (A–G) Images are confocal z-stacks (A and E) or single sections (B–D, F, and G). (A) Three-dimensional reconstruction of a live NRK cell nucleus expressing POM121-EGFP3. Top, maximum intensity projected of optical sections of the lower nuclear surface (double-pointed arrow). Inset enlarges 1 μm2 to show labeling of single NPCs. Bottom, xz slice at the line indicated in the top panel. (B) Live PtK2 cell coexpressing POM121-YFP3 (red) and SRβ-ECFP (green). AL labeled by POM121 are in direct contact to or colocalize with ER tubules and sheets as marked by SRβ. (C) HeLa cell expressing POM121-EGFP3 (green), fixed and stained for the endogenous nucleoporin p62 (red). Since the p62 antibody did not cross-react in PtK2 cells, HeLa cells were used for this experiment. AL containing POM121 colocalize with p62 antibodies (yellow in the merged lower panel, compare patterns in the split inset images). (D) Live PtK2 cell coexpressing ECFP–lamin B1 (green) and POM121-YFP3 (red). AL labeled by POM121 do not contain lamin B1. (E) Three-dimensional reconstruction of a live NRK cell nucleus expressing EGFP3-Nup153. Projections as in A. (F) Live COS7 cell coexpressing EGFP3-Nup153 (red) and SRβ-ECFP (green). COS7 cells were used for better ER morphology than NRK cells (compare with B). Yellow shows colocalization of AL with ER tubules. (G) COS7 cell expressing EGFP3-Nup153 (red) fixed and stained for the en-dogenous nucleoporin POM121 (red). AL containing Nup153 also label with anti-POM121 antibodies (yellow in the merged lower panel, compare patterns in the split inset images). Some POM121-positive structures do not contain Nup153. (H and I) Cryoimmuno double labeling electron micrographs from HeLa cells expressing POM121-EGFP3. Membrane boundaries are outlined next to the micrographs. Labeled are rat POM121 (10-nm gold) and human p62 (5-nm gold). POM121 and p62 colocalize on fenestrated membrane stacks (AL) in H and nuclear pores in I. M, mitochondria; N, nucleus; C, cytoplasm. Bars: (A–G) 5 μm; (H and I) 200 nm.
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fig1: POM121-GFP and GFP-Nup153 label single NPCs and induce AL in live cells. (A–G) Images are confocal z-stacks (A and E) or single sections (B–D, F, and G). (A) Three-dimensional reconstruction of a live NRK cell nucleus expressing POM121-EGFP3. Top, maximum intensity projected of optical sections of the lower nuclear surface (double-pointed arrow). Inset enlarges 1 μm2 to show labeling of single NPCs. Bottom, xz slice at the line indicated in the top panel. (B) Live PtK2 cell coexpressing POM121-YFP3 (red) and SRβ-ECFP (green). AL labeled by POM121 are in direct contact to or colocalize with ER tubules and sheets as marked by SRβ. (C) HeLa cell expressing POM121-EGFP3 (green), fixed and stained for the endogenous nucleoporin p62 (red). Since the p62 antibody did not cross-react in PtK2 cells, HeLa cells were used for this experiment. AL containing POM121 colocalize with p62 antibodies (yellow in the merged lower panel, compare patterns in the split inset images). (D) Live PtK2 cell coexpressing ECFP–lamin B1 (green) and POM121-YFP3 (red). AL labeled by POM121 do not contain lamin B1. (E) Three-dimensional reconstruction of a live NRK cell nucleus expressing EGFP3-Nup153. Projections as in A. (F) Live COS7 cell coexpressing EGFP3-Nup153 (red) and SRβ-ECFP (green). COS7 cells were used for better ER morphology than NRK cells (compare with B). Yellow shows colocalization of AL with ER tubules. (G) COS7 cell expressing EGFP3-Nup153 (red) fixed and stained for the en-dogenous nucleoporin POM121 (red). AL containing Nup153 also label with anti-POM121 antibodies (yellow in the merged lower panel, compare patterns in the split inset images). Some POM121-positive structures do not contain Nup153. (H and I) Cryoimmuno double labeling electron micrographs from HeLa cells expressing POM121-EGFP3. Membrane boundaries are outlined next to the micrographs. Labeled are rat POM121 (10-nm gold) and human p62 (5-nm gold). POM121 and p62 colocalize on fenestrated membrane stacks (AL) in H and nuclear pores in I. M, mitochondria; N, nucleus; C, cytoplasm. Bars: (A–G) 5 μm; (H and I) 200 nm.

Mentions: In this study we have tagged two well-characterized nucleoporins, POM121 (Hallberg et al., 1993) and Nup153 (Sukegawa and Blobel, 1993) with multiple copies (Zaal et al., 1999) of green fluorescent protein (GFP) to visualize them at low, nontoxic expression levels. Fusions of POM121 and Nup153 to single GFPs have been shown to incorporate into the NPC (Söderqvist et al., 1997; Pante et al., 2000). POM121-EG/YFP3 and EGFP2/3-Nup153 specifically labeled NPCs and were indistinguishable from the single-tagged proteins in all aspects except brightness. NPC number or distribution was not changed by expression of these chimeras as shown by high resolution confocal microscopy. We found an NPC density of ∼3.5 ± 0.5/μm2 and total number of NPCs per nucleus of 1660 ± 190 (n = 5; Fig. 1, A and E) . This was identical to NPCs counted in untransfected NRK cells by immunofluorescence using mAb 414 or anti-p62 antibodies (data not shown) and is in the range reported previously by ultrastructural and immunofluorescence analysis (Maul et al., 1980; Kubitscheck et al., 1996).


Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells.

Daigle N, Beaudouin J, Hartnell L, Imreh G, Hallberg E, Lippincott-Schwartz J, Ellenberg J - J. Cell Biol. (2001)

POM121-GFP and GFP-Nup153 label single NPCs and induce AL in live cells. (A–G) Images are confocal z-stacks (A and E) or single sections (B–D, F, and G). (A) Three-dimensional reconstruction of a live NRK cell nucleus expressing POM121-EGFP3. Top, maximum intensity projected of optical sections of the lower nuclear surface (double-pointed arrow). Inset enlarges 1 μm2 to show labeling of single NPCs. Bottom, xz slice at the line indicated in the top panel. (B) Live PtK2 cell coexpressing POM121-YFP3 (red) and SRβ-ECFP (green). AL labeled by POM121 are in direct contact to or colocalize with ER tubules and sheets as marked by SRβ. (C) HeLa cell expressing POM121-EGFP3 (green), fixed and stained for the endogenous nucleoporin p62 (red). Since the p62 antibody did not cross-react in PtK2 cells, HeLa cells were used for this experiment. AL containing POM121 colocalize with p62 antibodies (yellow in the merged lower panel, compare patterns in the split inset images). (D) Live PtK2 cell coexpressing ECFP–lamin B1 (green) and POM121-YFP3 (red). AL labeled by POM121 do not contain lamin B1. (E) Three-dimensional reconstruction of a live NRK cell nucleus expressing EGFP3-Nup153. Projections as in A. (F) Live COS7 cell coexpressing EGFP3-Nup153 (red) and SRβ-ECFP (green). COS7 cells were used for better ER morphology than NRK cells (compare with B). Yellow shows colocalization of AL with ER tubules. (G) COS7 cell expressing EGFP3-Nup153 (red) fixed and stained for the en-dogenous nucleoporin POM121 (red). AL containing Nup153 also label with anti-POM121 antibodies (yellow in the merged lower panel, compare patterns in the split inset images). Some POM121-positive structures do not contain Nup153. (H and I) Cryoimmuno double labeling electron micrographs from HeLa cells expressing POM121-EGFP3. Membrane boundaries are outlined next to the micrographs. Labeled are rat POM121 (10-nm gold) and human p62 (5-nm gold). POM121 and p62 colocalize on fenestrated membrane stacks (AL) in H and nuclear pores in I. M, mitochondria; N, nucleus; C, cytoplasm. Bars: (A–G) 5 μm; (H and I) 200 nm.
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fig1: POM121-GFP and GFP-Nup153 label single NPCs and induce AL in live cells. (A–G) Images are confocal z-stacks (A and E) or single sections (B–D, F, and G). (A) Three-dimensional reconstruction of a live NRK cell nucleus expressing POM121-EGFP3. Top, maximum intensity projected of optical sections of the lower nuclear surface (double-pointed arrow). Inset enlarges 1 μm2 to show labeling of single NPCs. Bottom, xz slice at the line indicated in the top panel. (B) Live PtK2 cell coexpressing POM121-YFP3 (red) and SRβ-ECFP (green). AL labeled by POM121 are in direct contact to or colocalize with ER tubules and sheets as marked by SRβ. (C) HeLa cell expressing POM121-EGFP3 (green), fixed and stained for the endogenous nucleoporin p62 (red). Since the p62 antibody did not cross-react in PtK2 cells, HeLa cells were used for this experiment. AL containing POM121 colocalize with p62 antibodies (yellow in the merged lower panel, compare patterns in the split inset images). (D) Live PtK2 cell coexpressing ECFP–lamin B1 (green) and POM121-YFP3 (red). AL labeled by POM121 do not contain lamin B1. (E) Three-dimensional reconstruction of a live NRK cell nucleus expressing EGFP3-Nup153. Projections as in A. (F) Live COS7 cell coexpressing EGFP3-Nup153 (red) and SRβ-ECFP (green). COS7 cells were used for better ER morphology than NRK cells (compare with B). Yellow shows colocalization of AL with ER tubules. (G) COS7 cell expressing EGFP3-Nup153 (red) fixed and stained for the en-dogenous nucleoporin POM121 (red). AL containing Nup153 also label with anti-POM121 antibodies (yellow in the merged lower panel, compare patterns in the split inset images). Some POM121-positive structures do not contain Nup153. (H and I) Cryoimmuno double labeling electron micrographs from HeLa cells expressing POM121-EGFP3. Membrane boundaries are outlined next to the micrographs. Labeled are rat POM121 (10-nm gold) and human p62 (5-nm gold). POM121 and p62 colocalize on fenestrated membrane stacks (AL) in H and nuclear pores in I. M, mitochondria; N, nucleus; C, cytoplasm. Bars: (A–G) 5 μm; (H and I) 200 nm.
Mentions: In this study we have tagged two well-characterized nucleoporins, POM121 (Hallberg et al., 1993) and Nup153 (Sukegawa and Blobel, 1993) with multiple copies (Zaal et al., 1999) of green fluorescent protein (GFP) to visualize them at low, nontoxic expression levels. Fusions of POM121 and Nup153 to single GFPs have been shown to incorporate into the NPC (Söderqvist et al., 1997; Pante et al., 2000). POM121-EG/YFP3 and EGFP2/3-Nup153 specifically labeled NPCs and were indistinguishable from the single-tagged proteins in all aspects except brightness. NPC number or distribution was not changed by expression of these chimeras as shown by high resolution confocal microscopy. We found an NPC density of ∼3.5 ± 0.5/μm2 and total number of NPCs per nucleus of 1660 ± 190 (n = 5; Fig. 1, A and E) . This was identical to NPCs counted in untransfected NRK cells by immunofluorescence using mAb 414 or anti-p62 antibodies (data not shown) and is in the range reported previously by ultrastructural and immunofluorescence analysis (Maul et al., 1980; Kubitscheck et al., 1996).

Bottom Line: No independent movement of single pore complexes was found within the plane of the NE in interphase.During mitosis, POM121 and Nup153 were completely dispersed and mobile in the ER (POM121) or cytosol (Nup153) in metaphase, and rapidly redistributed to an immobilized pool around chromatin in late anaphase.Assembly and immobilization of both nucleoporins occurred before detectable recruitment of lamin B1, which is thus unlikely to mediate initiation of NPC assembly at the end of mitosis.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.

ABSTRACT
The nuclear pore complex (NPC) and its relationship to the nuclear envelope (NE) was characterized in living cells using POM121-green fluorescent protein (GFP) and GFP-Nup153, and GFP-lamin B1. No independent movement of single pore complexes was found within the plane of the NE in interphase. Only large arrays of NPCs moved slowly and synchronously during global changes in nuclear shape, strongly suggesting mechanical connections which form an NPC network. The nuclear lamina exhibited identical movements. NPC turnover measured by fluorescence recovery after photobleaching of POM121 was less than once per cell cycle. Nup153 association with NPCs was dynamic and turnover of this nucleoporin was three orders of magnitude faster. Overexpression of both nucleoporins induced the formation of annulate lamellae (AL) in the endoplasmic reticulum (ER). Turnover of AL pore complexes was much higher than in the NE (once every 2.5 min). During mitosis, POM121 and Nup153 were completely dispersed and mobile in the ER (POM121) or cytosol (Nup153) in metaphase, and rapidly redistributed to an immobilized pool around chromatin in late anaphase. Assembly and immobilization of both nucleoporins occurred before detectable recruitment of lamin B1, which is thus unlikely to mediate initiation of NPC assembly at the end of mitosis.

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