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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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Recruitment of nucleoporins and lamin B1 during nuclear assembly. (A) In vivo localization of transiently expressed POM121-YFP3, EGFP3-Nup153, and ECFP–lamin B1 in metaphase NRK cells. Shown are confocal sections and DIC images centered on the metaphase plate. Note reticular pattern for POM121 and diffuse distribution of Nup153 and lamin B1. (B) 4-D sequence (10 slices every 2.5 μm acquired every 10–60 s depending on the dynamics of the cell cycle stage) of an NRK cell transiently expressing EGFP2-Nup153. Transparent projection of the four slices containing chromosomes (top) and DIC images highlighting the position of the chromosomes (bottom) are shown. Recruitment of Nup153 on the surface of chromosomes starts at ∼4 min after metaphase to anaphase transition. Time, h:mm:ss normalized to meta/anaphase transition equals 0. (C) 4-D double label sequence (six slices every 3 μm acquired every 30–120 s depending on the dynamics of the cell cycle stage) of an NRK cell transiently coexpressing POM121-YFP3 (top) and ECFP–lamin B1 (bottom). Shown are maximum intensity projections of the z-slices containing chromosomes. POM121 recruitment starts ∼3.5 min after meta/anaphase transition, whereas lamin B1 is only seen at 9 min (see D). Time, h:mm:ss normalized to meta/anaphase transition equals 0. (D) Plot of mean fluorescence intensity of the reforming NE for POM121 (green) and lamin B1 (black) shown in B. Before visible accumulation of lamin, nuclear rim areas were identified by POM121 localization. Data was background subtracted and normalized for bleaching during the time series. Lines are moving averages of two frames. Note the delay of ∼5 min between peak concentration/area for POM121 versus lamin B1. Bars: (A) 5 μm; (B and C) 10 μm. Online supplemental material (Fig. S2) is available at http://www.jcb.org/cgi/content/full/200101089/DC1.
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fig5: Recruitment of nucleoporins and lamin B1 during nuclear assembly. (A) In vivo localization of transiently expressed POM121-YFP3, EGFP3-Nup153, and ECFP–lamin B1 in metaphase NRK cells. Shown are confocal sections and DIC images centered on the metaphase plate. Note reticular pattern for POM121 and diffuse distribution of Nup153 and lamin B1. (B) 4-D sequence (10 slices every 2.5 μm acquired every 10–60 s depending on the dynamics of the cell cycle stage) of an NRK cell transiently expressing EGFP2-Nup153. Transparent projection of the four slices containing chromosomes (top) and DIC images highlighting the position of the chromosomes (bottom) are shown. Recruitment of Nup153 on the surface of chromosomes starts at ∼4 min after metaphase to anaphase transition. Time, h:mm:ss normalized to meta/anaphase transition equals 0. (C) 4-D double label sequence (six slices every 3 μm acquired every 30–120 s depending on the dynamics of the cell cycle stage) of an NRK cell transiently coexpressing POM121-YFP3 (top) and ECFP–lamin B1 (bottom). Shown are maximum intensity projections of the z-slices containing chromosomes. POM121 recruitment starts ∼3.5 min after meta/anaphase transition, whereas lamin B1 is only seen at 9 min (see D). Time, h:mm:ss normalized to meta/anaphase transition equals 0. (D) Plot of mean fluorescence intensity of the reforming NE for POM121 (green) and lamin B1 (black) shown in B. Before visible accumulation of lamin, nuclear rim areas were identified by POM121 localization. Data was background subtracted and normalized for bleaching during the time series. Lines are moving averages of two frames. Note the delay of ∼5 min between peak concentration/area for POM121 versus lamin B1. Bars: (A) 5 μm; (B and C) 10 μm. Online supplemental material (Fig. S2) is available at http://www.jcb.org/cgi/content/full/200101089/DC1.

Mentions: Having characterized the dynamics of NPCs in interphase we next investigated their biogenesis after mitosis. Four-dimensional (4-D) confocal time-lapse microscopy was used to follow cells from metaphase to G1 expressing either POM121-YFP3, EGFP2-Nup153, or ECFP–lamin B1. No structures that could have reflected intact NPCs, ALs, or lamin filaments were detected in metaphase, consistent with the complete disassembly of nuclear structure in mitosis. POM121, a nuclear membrane protein, was found equilibrated with the ER, whereas both Nup153 and lamin B1 were dispersed homogeneously in the cytosol (Fig. 5 A). The nucleoporins POM121 and Nup153 started to concentrate around chromatin in anaphase B, ∼4 min after metaphase/anaphase transition, which served as a temporal reference to compare different cells (Fig. 5, B and C) at the same time as nuclear membrane assembly marked by LBR-GFP (data not shown; Ellenberg et al., 1997). POM121 and Nup153 rapidly concentrated around chromatin, depleting their ER/cytoplasmic pool almost entirely by the end of telophase (Fig. 5, B and C). ALs, which were present in the POM121-YFP3–expressing cells before mitosis, reformed only much later with the onset of cytokinesis (Fig. 5 C, top). Since NPCs appeared to be linked to the lamina in interphase nuclei, we reasoned that lamins might play an important role in pore complex assembly. To our surprise, confocal time-lapse analysis of coexpressed ECFP–lamin B1 and POM121-YFP3 revealed that lamin B1 recruitment to chromosomes occurred ∼5 min (Fig. 5, C and D) after POM121 recruitment. Concentration of lamin B1 around chromatin could only be detected in late telophase/early cytokinesis (Fig. 5, B and C), a stage when chromatin is already sealed by a pore containing membrane (Fig. 5, B and C). To confirm that the GFP-tagged proteins reflected the behavior of the endogenous proteins, double immunofluorescence of POM121 and lamins was performed in untransfected NRK cells. As seen in vivo, POM121 assembly took place in early anaphase, whereas lamins were recruited to chromatin only in telophase (see online supplemental Fig. S2, available at http://www.jcb.org/cgi/content/full/200101089/DC1).


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)

Recruitment of nucleoporins and lamin B1 during nuclear assembly. (A) In vivo localization of transiently expressed POM121-YFP3, EGFP3-Nup153, and ECFP–lamin B1 in metaphase NRK cells. Shown are confocal sections and DIC images centered on the metaphase plate. Note reticular pattern for POM121 and diffuse distribution of Nup153 and lamin B1. (B) 4-D sequence (10 slices every 2.5 μm acquired every 10–60 s depending on the dynamics of the cell cycle stage) of an NRK cell transiently expressing EGFP2-Nup153. Transparent projection of the four slices containing chromosomes (top) and DIC images highlighting the position of the chromosomes (bottom) are shown. Recruitment of Nup153 on the surface of chromosomes starts at ∼4 min after metaphase to anaphase transition. Time, h:mm:ss normalized to meta/anaphase transition equals 0. (C) 4-D double label sequence (six slices every 3 μm acquired every 30–120 s depending on the dynamics of the cell cycle stage) of an NRK cell transiently coexpressing POM121-YFP3 (top) and ECFP–lamin B1 (bottom). Shown are maximum intensity projections of the z-slices containing chromosomes. POM121 recruitment starts ∼3.5 min after meta/anaphase transition, whereas lamin B1 is only seen at 9 min (see D). Time, h:mm:ss normalized to meta/anaphase transition equals 0. (D) Plot of mean fluorescence intensity of the reforming NE for POM121 (green) and lamin B1 (black) shown in B. Before visible accumulation of lamin, nuclear rim areas were identified by POM121 localization. Data was background subtracted and normalized for bleaching during the time series. Lines are moving averages of two frames. Note the delay of ∼5 min between peak concentration/area for POM121 versus lamin B1. Bars: (A) 5 μm; (B and C) 10 μm. Online supplemental material (Fig. S2) is available at http://www.jcb.org/cgi/content/full/200101089/DC1.
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fig5: Recruitment of nucleoporins and lamin B1 during nuclear assembly. (A) In vivo localization of transiently expressed POM121-YFP3, EGFP3-Nup153, and ECFP–lamin B1 in metaphase NRK cells. Shown are confocal sections and DIC images centered on the metaphase plate. Note reticular pattern for POM121 and diffuse distribution of Nup153 and lamin B1. (B) 4-D sequence (10 slices every 2.5 μm acquired every 10–60 s depending on the dynamics of the cell cycle stage) of an NRK cell transiently expressing EGFP2-Nup153. Transparent projection of the four slices containing chromosomes (top) and DIC images highlighting the position of the chromosomes (bottom) are shown. Recruitment of Nup153 on the surface of chromosomes starts at ∼4 min after metaphase to anaphase transition. Time, h:mm:ss normalized to meta/anaphase transition equals 0. (C) 4-D double label sequence (six slices every 3 μm acquired every 30–120 s depending on the dynamics of the cell cycle stage) of an NRK cell transiently coexpressing POM121-YFP3 (top) and ECFP–lamin B1 (bottom). Shown are maximum intensity projections of the z-slices containing chromosomes. POM121 recruitment starts ∼3.5 min after meta/anaphase transition, whereas lamin B1 is only seen at 9 min (see D). Time, h:mm:ss normalized to meta/anaphase transition equals 0. (D) Plot of mean fluorescence intensity of the reforming NE for POM121 (green) and lamin B1 (black) shown in B. Before visible accumulation of lamin, nuclear rim areas were identified by POM121 localization. Data was background subtracted and normalized for bleaching during the time series. Lines are moving averages of two frames. Note the delay of ∼5 min between peak concentration/area for POM121 versus lamin B1. Bars: (A) 5 μm; (B and C) 10 μm. Online supplemental material (Fig. S2) is available at http://www.jcb.org/cgi/content/full/200101089/DC1.
Mentions: Having characterized the dynamics of NPCs in interphase we next investigated their biogenesis after mitosis. Four-dimensional (4-D) confocal time-lapse microscopy was used to follow cells from metaphase to G1 expressing either POM121-YFP3, EGFP2-Nup153, or ECFP–lamin B1. No structures that could have reflected intact NPCs, ALs, or lamin filaments were detected in metaphase, consistent with the complete disassembly of nuclear structure in mitosis. POM121, a nuclear membrane protein, was found equilibrated with the ER, whereas both Nup153 and lamin B1 were dispersed homogeneously in the cytosol (Fig. 5 A). The nucleoporins POM121 and Nup153 started to concentrate around chromatin in anaphase B, ∼4 min after metaphase/anaphase transition, which served as a temporal reference to compare different cells (Fig. 5, B and C) at the same time as nuclear membrane assembly marked by LBR-GFP (data not shown; Ellenberg et al., 1997). POM121 and Nup153 rapidly concentrated around chromatin, depleting their ER/cytoplasmic pool almost entirely by the end of telophase (Fig. 5, B and C). ALs, which were present in the POM121-YFP3–expressing cells before mitosis, reformed only much later with the onset of cytokinesis (Fig. 5 C, top). Since NPCs appeared to be linked to the lamina in interphase nuclei, we reasoned that lamins might play an important role in pore complex assembly. To our surprise, confocal time-lapse analysis of coexpressed ECFP–lamin B1 and POM121-YFP3 revealed that lamin B1 recruitment to chromosomes occurred ∼5 min (Fig. 5, C and D) after POM121 recruitment. Concentration of lamin B1 around chromatin could only be detected in late telophase/early cytokinesis (Fig. 5, B and C), a stage when chromatin is already sealed by a pore containing membrane (Fig. 5, B and C). To confirm that the GFP-tagged proteins reflected the behavior of the endogenous proteins, double immunofluorescence of POM121 and lamins was performed in untransfected NRK cells. As seen in vivo, POM121 assembly took place in early anaphase, whereas lamins were recruited to chromatin only in telophase (see online supplemental Fig. S2, available at http://www.jcb.org/cgi/content/full/200101089/DC1).

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.

Show MeSH