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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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Tracking of NPC and lamina movement in interphase. (A) Time-lapse of a NRK cell expressing POM121-EGFP3 transiently. The lower nuclear surface was followed in a single confocal section every 2 s for a total of 30 min on a real-time confocal microscope. Sequence was averaged with a running window of five frames. Representative frames show numbered NPCs in a time window of ∼2 min used for tracking in B. Time, mm:ss. NPC movement is difficult to appreciate in still images; see Video 3 for the complete sequence. (B) Tracking of NPC movement. NPCs labeled #1–5 in A in a region of local NE movement are tracked together with two NPCs labeled C1 and C2 in A, which reside in an area of little movement and serve to illustrate global nuclear drift. Note the parallel and synchronous tracks of the NPCs. (C) Pattern FRAP of an NRK cell nucleus transiently expressing EGFP–Lamin B1. 33 1 × 0.5-μm regions were photobleached in the lower nuclear surface. Movement of landmarks was followed in single confocal sections every minute for 30 min (top row). Marks in the boxed area were used to track elastic deformations of the lamin lattice. Global cellular movement was corrected with two reference points. Relative position changes are shown in the bottom row as a network connecting the center of the bleach marks. Time, h:mm:ss; horizontal box length, 6 μm. (D) Pattern FRAP of a NRK nucleus transiently coexpressing POM121-YFP3 and ECFP–lamin B1. The outlined 21 0.9 × 0.6-μm regions were photobleached selectively in the lamina using a 413-nm Kr laser line. Movement of the lamina landmarks and the unbleached NPCs was then followed in single double-labeled confocal sections every 31 s for 30 min. Representative frames show distortion of nuclear shape by cell migration (note lamina folding at 07:56 and 12:40). Marks and NPCs used for tracking in E are red. Time, mm:ss. See Video 4 to better appreciate lamina elasticity. (E) Tracking of NPCs and lamina bleachmarks. Exemplary time-space tracks are shown for NPC #1 (green) and lamin grid marks A1–B2 (black) over 22 min in x, t and y, t plots. Global nuclear drift was normalized using to B5 and B7 marks. Note correlation between the x and y lamina and NPC movement. NPCs #2 and 3 and the surrounding marks behaved identically (not shown). Bars, 5 μm. Online supplemental material (Videos 3 and 4) is available at http://www.jcb.org/cgi/content/full/200101089/DC1.
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fig4: Tracking of NPC and lamina movement in interphase. (A) Time-lapse of a NRK cell expressing POM121-EGFP3 transiently. The lower nuclear surface was followed in a single confocal section every 2 s for a total of 30 min on a real-time confocal microscope. Sequence was averaged with a running window of five frames. Representative frames show numbered NPCs in a time window of ∼2 min used for tracking in B. Time, mm:ss. NPC movement is difficult to appreciate in still images; see Video 3 for the complete sequence. (B) Tracking of NPC movement. NPCs labeled #1–5 in A in a region of local NE movement are tracked together with two NPCs labeled C1 and C2 in A, which reside in an area of little movement and serve to illustrate global nuclear drift. Note the parallel and synchronous tracks of the NPCs. (C) Pattern FRAP of an NRK cell nucleus transiently expressing EGFP–Lamin B1. 33 1 × 0.5-μm regions were photobleached in the lower nuclear surface. Movement of landmarks was followed in single confocal sections every minute for 30 min (top row). Marks in the boxed area were used to track elastic deformations of the lamin lattice. Global cellular movement was corrected with two reference points. Relative position changes are shown in the bottom row as a network connecting the center of the bleach marks. Time, h:mm:ss; horizontal box length, 6 μm. (D) Pattern FRAP of a NRK nucleus transiently coexpressing POM121-YFP3 and ECFP–lamin B1. The outlined 21 0.9 × 0.6-μm regions were photobleached selectively in the lamina using a 413-nm Kr laser line. Movement of the lamina landmarks and the unbleached NPCs was then followed in single double-labeled confocal sections every 31 s for 30 min. Representative frames show distortion of nuclear shape by cell migration (note lamina folding at 07:56 and 12:40). Marks and NPCs used for tracking in E are red. Time, mm:ss. See Video 4 to better appreciate lamina elasticity. (E) Tracking of NPCs and lamina bleachmarks. Exemplary time-space tracks are shown for NPC #1 (green) and lamin grid marks A1–B2 (black) over 22 min in x, t and y, t plots. Global nuclear drift was normalized using to B5 and B7 marks. Note correlation between the x and y lamina and NPC movement. NPCs #2 and 3 and the surrounding marks behaved identically (not shown). Bars, 5 μm. Online supplemental material (Videos 3 and 4) is available at http://www.jcb.org/cgi/content/full/200101089/DC1.

Mentions: FRAP experiments of POM121 also revealed a stable boundary between bleached and nonbleached nuclear regions suggesting individual NPCs undergo little independent movement. (Figs. 2 A and 3 A). To investigate this further, we performed time-lapse imaging of the lower nuclear surface at single pore resolution (Fig. 4 A and Video 3). Tracking sets of individual NPCs showed no independent movement (Fig. 4 B). Rather, large arrays of NPCs moved in synchronous waves and the relative position of individual NPCs remained constant (Fig. 4 A and Video 3). This behavior was consistent with a spatially constrained two-dimensional network of pores. The slowly turned over lamina (Fig. 2, B and C) was a good candidate to connect mechanically such a network. To investigate this, double label time-lapse tracking experiments with NRK cells coexpressing ECFP–lamin B1 and POM121-YFP3 were performed. To track movement on the smooth surface of the lamina, we selectively photobleached a pattern of 21 0.9 × 0.6 μm landmarks into the ECFP-lamin B1–labeled lamina and followed movements of NPCs and the lamin pattern for 30 min (Fig. 4, D and E). NPC and lamin movement were strictly correlated, consistent with lamins and NPCs being part of the same network. That this network was elastic became evident in landmark-tracking experiments on migrating cells expressing only EGFP-lamin B1 (Fig. 4 C and Video 4). Folds passing through the lower nuclear surface distorted the grid only temporarily. The grid always relaxed back to its original position at the end of cellular movement (Fig. 4 C and Video 4). Supplemental videos are 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)

Tracking of NPC and lamina movement in interphase. (A) Time-lapse of a NRK cell expressing POM121-EGFP3 transiently. The lower nuclear surface was followed in a single confocal section every 2 s for a total of 30 min on a real-time confocal microscope. Sequence was averaged with a running window of five frames. Representative frames show numbered NPCs in a time window of ∼2 min used for tracking in B. Time, mm:ss. NPC movement is difficult to appreciate in still images; see Video 3 for the complete sequence. (B) Tracking of NPC movement. NPCs labeled #1–5 in A in a region of local NE movement are tracked together with two NPCs labeled C1 and C2 in A, which reside in an area of little movement and serve to illustrate global nuclear drift. Note the parallel and synchronous tracks of the NPCs. (C) Pattern FRAP of an NRK cell nucleus transiently expressing EGFP–Lamin B1. 33 1 × 0.5-μm regions were photobleached in the lower nuclear surface. Movement of landmarks was followed in single confocal sections every minute for 30 min (top row). Marks in the boxed area were used to track elastic deformations of the lamin lattice. Global cellular movement was corrected with two reference points. Relative position changes are shown in the bottom row as a network connecting the center of the bleach marks. Time, h:mm:ss; horizontal box length, 6 μm. (D) Pattern FRAP of a NRK nucleus transiently coexpressing POM121-YFP3 and ECFP–lamin B1. The outlined 21 0.9 × 0.6-μm regions were photobleached selectively in the lamina using a 413-nm Kr laser line. Movement of the lamina landmarks and the unbleached NPCs was then followed in single double-labeled confocal sections every 31 s for 30 min. Representative frames show distortion of nuclear shape by cell migration (note lamina folding at 07:56 and 12:40). Marks and NPCs used for tracking in E are red. Time, mm:ss. See Video 4 to better appreciate lamina elasticity. (E) Tracking of NPCs and lamina bleachmarks. Exemplary time-space tracks are shown for NPC #1 (green) and lamin grid marks A1–B2 (black) over 22 min in x, t and y, t plots. Global nuclear drift was normalized using to B5 and B7 marks. Note correlation between the x and y lamina and NPC movement. NPCs #2 and 3 and the surrounding marks behaved identically (not shown). Bars, 5 μm. Online supplemental material (Videos 3 and 4) is available at http://www.jcb.org/cgi/content/full/200101089/DC1.
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fig4: Tracking of NPC and lamina movement in interphase. (A) Time-lapse of a NRK cell expressing POM121-EGFP3 transiently. The lower nuclear surface was followed in a single confocal section every 2 s for a total of 30 min on a real-time confocal microscope. Sequence was averaged with a running window of five frames. Representative frames show numbered NPCs in a time window of ∼2 min used for tracking in B. Time, mm:ss. NPC movement is difficult to appreciate in still images; see Video 3 for the complete sequence. (B) Tracking of NPC movement. NPCs labeled #1–5 in A in a region of local NE movement are tracked together with two NPCs labeled C1 and C2 in A, which reside in an area of little movement and serve to illustrate global nuclear drift. Note the parallel and synchronous tracks of the NPCs. (C) Pattern FRAP of an NRK cell nucleus transiently expressing EGFP–Lamin B1. 33 1 × 0.5-μm regions were photobleached in the lower nuclear surface. Movement of landmarks was followed in single confocal sections every minute for 30 min (top row). Marks in the boxed area were used to track elastic deformations of the lamin lattice. Global cellular movement was corrected with two reference points. Relative position changes are shown in the bottom row as a network connecting the center of the bleach marks. Time, h:mm:ss; horizontal box length, 6 μm. (D) Pattern FRAP of a NRK nucleus transiently coexpressing POM121-YFP3 and ECFP–lamin B1. The outlined 21 0.9 × 0.6-μm regions were photobleached selectively in the lamina using a 413-nm Kr laser line. Movement of the lamina landmarks and the unbleached NPCs was then followed in single double-labeled confocal sections every 31 s for 30 min. Representative frames show distortion of nuclear shape by cell migration (note lamina folding at 07:56 and 12:40). Marks and NPCs used for tracking in E are red. Time, mm:ss. See Video 4 to better appreciate lamina elasticity. (E) Tracking of NPCs and lamina bleachmarks. Exemplary time-space tracks are shown for NPC #1 (green) and lamin grid marks A1–B2 (black) over 22 min in x, t and y, t plots. Global nuclear drift was normalized using to B5 and B7 marks. Note correlation between the x and y lamina and NPC movement. NPCs #2 and 3 and the surrounding marks behaved identically (not shown). Bars, 5 μm. Online supplemental material (Videos 3 and 4) is available at http://www.jcb.org/cgi/content/full/200101089/DC1.
Mentions: FRAP experiments of POM121 also revealed a stable boundary between bleached and nonbleached nuclear regions suggesting individual NPCs undergo little independent movement. (Figs. 2 A and 3 A). To investigate this further, we performed time-lapse imaging of the lower nuclear surface at single pore resolution (Fig. 4 A and Video 3). Tracking sets of individual NPCs showed no independent movement (Fig. 4 B). Rather, large arrays of NPCs moved in synchronous waves and the relative position of individual NPCs remained constant (Fig. 4 A and Video 3). This behavior was consistent with a spatially constrained two-dimensional network of pores. The slowly turned over lamina (Fig. 2, B and C) was a good candidate to connect mechanically such a network. To investigate this, double label time-lapse tracking experiments with NRK cells coexpressing ECFP–lamin B1 and POM121-YFP3 were performed. To track movement on the smooth surface of the lamina, we selectively photobleached a pattern of 21 0.9 × 0.6 μm landmarks into the ECFP-lamin B1–labeled lamina and followed movements of NPCs and the lamin pattern for 30 min (Fig. 4, D and E). NPC and lamin movement were strictly correlated, consistent with lamins and NPCs being part of the same network. That this network was elastic became evident in landmark-tracking experiments on migrating cells expressing only EGFP-lamin B1 (Fig. 4 C and Video 4). Folds passing through the lower nuclear surface distorted the grid only temporarily. The grid always relaxed back to its original position at the end of cellular movement (Fig. 4 C and Video 4). Supplemental videos are 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
Related in: MedlinePlus