Limits...
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.

Show MeSH
Turnover of nucleoporins within NPCs and ALPCs measured by FRAP. (A) FRAP of NRK cells expressing POM121-EGFP3 transiently. Boxed regions were photobleached and fluorescence recovery of ALPCs and NPCs was followed immediately after the bleach and then every 28 s in confocal sections. Arrowheads indicate the bleached AL. Note difference in recovery of AL and NPCs. Time, mm:ss. For quantitation see online supplemental Fig. S1 A. (B) FRAP of NRK cells expressing EGFP2-Nup153 transiently. Recovery was monitored immediately after the bleach and then every 5 s. Time and scale as in A. (C) FLIP of PtK2 cells expressing EGFP2-Nup153 transiently. Outlined region was photobleached repetitively 30 times every 30 s. Before and after each bleach the depletion of fluorescence was monitored in a confocal section. Time and scale as in A. (D) Plots of recovery in the bleached half and equilibration between bleached and nonbleached half of a Nup153 FRAP similar to B. Average mean intensities of the bleached region (black, left Y-axis) and standard deviation (n = 4). Data was normalized for total loss of fluorescence. Change of total fluorescence from the nonbleached (green) and bleached half (blue, both right Y-axis) measured for the experiment shown in B. (E) Plots of EGFP2-Nup153 depletion from the nucleus and cytoplasm for the FLIP in C. Average nuclear (black), cytoplasmic (green, both left Y-axis), and ratio of NPC/cytoplasmic fluorescence (blue, right Y-axis) is shown. Data was normalized to total loss of fluorescence. Note that nuclear fluorescence remains constant after initial loss of overlapping cytoplasmic signal, whereas cytoplasmic fluorescence is reduced to background levels. Bars, 5 μm. Online supplemental material (Fig. S1) is available at http://www.jcb.org/cgi/content/full/200101089/DC1.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196857&req=5

fig3: Turnover of nucleoporins within NPCs and ALPCs measured by FRAP. (A) FRAP of NRK cells expressing POM121-EGFP3 transiently. Boxed regions were photobleached and fluorescence recovery of ALPCs and NPCs was followed immediately after the bleach and then every 28 s in confocal sections. Arrowheads indicate the bleached AL. Note difference in recovery of AL and NPCs. Time, mm:ss. For quantitation see online supplemental Fig. S1 A. (B) FRAP of NRK cells expressing EGFP2-Nup153 transiently. Recovery was monitored immediately after the bleach and then every 5 s. Time and scale as in A. (C) FLIP of PtK2 cells expressing EGFP2-Nup153 transiently. Outlined region was photobleached repetitively 30 times every 30 s. Before and after each bleach the depletion of fluorescence was monitored in a confocal section. Time and scale as in A. (D) Plots of recovery in the bleached half and equilibration between bleached and nonbleached half of a Nup153 FRAP similar to B. Average mean intensities of the bleached region (black, left Y-axis) and standard deviation (n = 4). Data was normalized for total loss of fluorescence. Change of total fluorescence from the nonbleached (green) and bleached half (blue, both right Y-axis) measured for the experiment shown in B. (E) Plots of EGFP2-Nup153 depletion from the nucleus and cytoplasm for the FLIP in C. Average nuclear (black), cytoplasmic (green, both left Y-axis), and ratio of NPC/cytoplasmic fluorescence (blue, right Y-axis) is shown. Data was normalized to total loss of fluorescence. Note that nuclear fluorescence remains constant after initial loss of overlapping cytoplasmic signal, whereas cytoplasmic fluorescence is reduced to background levels. Bars, 5 μm. Online supplemental material (Fig. S1) is available at http://www.jcb.org/cgi/content/full/200101089/DC1.

Mentions: The low turnover of the NPC in the nuclear membrane prompted us to investigate pore complex turnover in AL by FRAP analysis, since ALPCs appeared similar to NPCs by cryoimmunoelectron microscopy (Fig. 1, H and I). A significant portion of AL associated POM121-EGFP3 exchanged completely after only ∼8 min, with a t1/2 of ∼76 ± 15 s (n = 6), i.e., three orders of magnitude faster than in NPCs, which showed no exchange during this time (Fig. 3 A and supplemental data). However, there was a significant IF of AL-associated POM121 varying between 50–70% (see online supplemental Fig. S1, 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)

Turnover of nucleoporins within NPCs and ALPCs measured by FRAP. (A) FRAP of NRK cells expressing POM121-EGFP3 transiently. Boxed regions were photobleached and fluorescence recovery of ALPCs and NPCs was followed immediately after the bleach and then every 28 s in confocal sections. Arrowheads indicate the bleached AL. Note difference in recovery of AL and NPCs. Time, mm:ss. For quantitation see online supplemental Fig. S1 A. (B) FRAP of NRK cells expressing EGFP2-Nup153 transiently. Recovery was monitored immediately after the bleach and then every 5 s. Time and scale as in A. (C) FLIP of PtK2 cells expressing EGFP2-Nup153 transiently. Outlined region was photobleached repetitively 30 times every 30 s. Before and after each bleach the depletion of fluorescence was monitored in a confocal section. Time and scale as in A. (D) Plots of recovery in the bleached half and equilibration between bleached and nonbleached half of a Nup153 FRAP similar to B. Average mean intensities of the bleached region (black, left Y-axis) and standard deviation (n = 4). Data was normalized for total loss of fluorescence. Change of total fluorescence from the nonbleached (green) and bleached half (blue, both right Y-axis) measured for the experiment shown in B. (E) Plots of EGFP2-Nup153 depletion from the nucleus and cytoplasm for the FLIP in C. Average nuclear (black), cytoplasmic (green, both left Y-axis), and ratio of NPC/cytoplasmic fluorescence (blue, right Y-axis) is shown. Data was normalized to total loss of fluorescence. Note that nuclear fluorescence remains constant after initial loss of overlapping cytoplasmic signal, whereas cytoplasmic fluorescence is reduced to background levels. Bars, 5 μm. Online supplemental material (Fig. S1) is available at http://www.jcb.org/cgi/content/full/200101089/DC1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196857&req=5

fig3: Turnover of nucleoporins within NPCs and ALPCs measured by FRAP. (A) FRAP of NRK cells expressing POM121-EGFP3 transiently. Boxed regions were photobleached and fluorescence recovery of ALPCs and NPCs was followed immediately after the bleach and then every 28 s in confocal sections. Arrowheads indicate the bleached AL. Note difference in recovery of AL and NPCs. Time, mm:ss. For quantitation see online supplemental Fig. S1 A. (B) FRAP of NRK cells expressing EGFP2-Nup153 transiently. Recovery was monitored immediately after the bleach and then every 5 s. Time and scale as in A. (C) FLIP of PtK2 cells expressing EGFP2-Nup153 transiently. Outlined region was photobleached repetitively 30 times every 30 s. Before and after each bleach the depletion of fluorescence was monitored in a confocal section. Time and scale as in A. (D) Plots of recovery in the bleached half and equilibration between bleached and nonbleached half of a Nup153 FRAP similar to B. Average mean intensities of the bleached region (black, left Y-axis) and standard deviation (n = 4). Data was normalized for total loss of fluorescence. Change of total fluorescence from the nonbleached (green) and bleached half (blue, both right Y-axis) measured for the experiment shown in B. (E) Plots of EGFP2-Nup153 depletion from the nucleus and cytoplasm for the FLIP in C. Average nuclear (black), cytoplasmic (green, both left Y-axis), and ratio of NPC/cytoplasmic fluorescence (blue, right Y-axis) is shown. Data was normalized to total loss of fluorescence. Note that nuclear fluorescence remains constant after initial loss of overlapping cytoplasmic signal, whereas cytoplasmic fluorescence is reduced to background levels. Bars, 5 μm. Online supplemental material (Fig. S1) is available at http://www.jcb.org/cgi/content/full/200101089/DC1.
Mentions: The low turnover of the NPC in the nuclear membrane prompted us to investigate pore complex turnover in AL by FRAP analysis, since ALPCs appeared similar to NPCs by cryoimmunoelectron microscopy (Fig. 1, H and I). A significant portion of AL associated POM121-EGFP3 exchanged completely after only ∼8 min, with a t1/2 of ∼76 ± 15 s (n = 6), i.e., three orders of magnitude faster than in NPCs, which showed no exchange during this time (Fig. 3 A and supplemental data). However, there was a significant IF of AL-associated POM121 varying between 50–70% (see online supplemental Fig. S1, 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